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Abstract Autophagy is activated in response to a variety of stress conditions including anti-cancer therapies, and tumors cells often depend on autophagy for survival. In this study, we have evaluated inhibition of autophagy as therapeutic strategy in acute lymphoblastic leukemia (ALL) in children, both as a single treatment and in combination with glucocorticoid (GC) Dexamethasone (Dexa). Analysis of proteomics and RNA-seq of ALL cell lines and primary samples identified an upregulation of Vps34 and ATG14 proteins and autophagy and lysosomal pathway enrichment in a genetic subgroup with a recurrent t(12;21) translocation. Cells from this sugbroup were also significantly more sensitive to the selective autophagy or lysosomal inhibitors than cells with other genetic rearrangements. Further, combination of Dexa with either lysosomal or autophagy inhibitors was either synergistic or additive in killing leukemic cells across various genetic and lineage backgrounds, for both cell lines and primary samples, as assessed using viability assays and SynergyFinder as well as apoptotic caspase 3/7-based live-cell assays. Our data demonstrate that targeting autophagy represents a promising strategy for the treatment of pediatric ALL, both as a selective modality for the t(12;21) pre-B-ALL subgroup, and in combination treatments to sensitize to GC-induced cytotoxicity.

Abstract Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142− /low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142− /low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Abstract Background Bronchopulmonary Dysplasia (BPD) in infants born prematurely is a risk factor for chronic airway obstruction later in life. The distribution of T cell subtypes in the large airways is largely unknown. Objective To characterize cellular and T cell profiles in the large airways of young adults with a history of BPD. Methods Forty-three young adults born prematurely (preterm (n = 20), BPD (n = 23)) and 45 full-term-born (asthma (n = 23), healthy (n = 22)) underwent lung function measurements, and bronchoscopy with large airway bronchial wash (BW). T-cells subsets in BW were analyzed by immunocytochemistry. Results The proportions of both lymphocytes and CD8 + T cells in BW were significantly higher in BPD (median, 6.6%, and 78.0%) when compared with asthma (3.4% and 67.8%, p = 0.002 and p = 0.040) and healthy (3.8% and 40%, p

IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA).MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up.ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value

In classical Hodgkin lymphoma (cHL), the malignant cells represent only a small fraction of the tumor. Yet, they orchestrate a lymphocyte-dominated tumor microenvironment (TME) that supports their survival and growth. The systemic effects of this local immunomodulation are not fully elucidated. Here, we aimed at characterizing circulating lymphocytes and plasma proteins in relation to clinical parameters and treatment effect. Peripheral blood (PB) samples were obtained from 48 consecutive patients at diagnosis and at 2 time points after successful primary treatment. Single-cell suspensions were prepared from lymph node (LN) biopsies obtained for routine diagnostic purposes. Twenty healthy individuals were included as controls. Cells from PB and LN were analyzed by flow cytometry, and plasma proteins by Proximity Extension Assay. We found that the frequencies of T and B cells positively correlated between the LN and the PB compartments. Compared to controls, cHL patients had higher frequencies of proliferating T cells as well as higher expression of programmed death (PD)-1 and cytotoxic T lymphocyte antigen (CTLA)-4 in circulating T cells, and lower naive T-cell frequencies. Advanced-stage patients had fewer NK cells with a functionally impaired phenotype. Differences in the immune profile were observed in patients with a high tumor burden and with high inflammation, respectively. Most of these deviations disappeared after standard first-line treatment. Patients who received radiotherapy involving the mediastinum had low T-cell counts for a prolonged period. Our findings suggest that the immunomodulation of lymphocytes in the TME of cHL might affect immune biomarkers in the PB.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Gene and protein expression of programmed death‐ligand 1 (PD‐L1) are prognostic in early breast cancer (BC), but their prognostic information is inconsistent at least in some biological subgroups. The validated prognostic gene signatures (GS) in BC are mainly based on proliferation and estrogen receptor (ER)‐related genes. Here, we aimed to explore the prognostic capacity of PD‐L1 expression at the protein vs mRNA levels and to investigate the prognostic information that PD‐L1 can potentially add to routinely used GS. Gene expression data were derived from two early BC cohorts (cohort 1: 562 patients; cohort 2: 1081 patients). Tissue microarrays from cohort 1 were immunohistochemically (IHC) stained for PD‐L1 using the SP263 clone. GS scores (21‐gene, 70‐gene) were calculated, and likelihood‐ratio (LR) tests and concordance indices were used to evaluate the additional prognostic information for each signature. The immune cell composition was also evaluated using the CIBERSORT in silico tool. PD‐L1 gene and protein expressions were independently associated with better prognosis. In ER+/HER2− patients, PD‐L1 gene expression provided significant additional prognostic information beyond that of both 21‐GS [LR‐Δχ2 = 15.289 and LR‐Δχ2 = 8.812, P

Abstract The deoxycytidine analogue cytarabine (ara‐C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara‐C efficacy by hydrolysing the active triphosphate metabolite ara‐CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1‐mediated barrier to ara‐C efficacy in primary blasts and mouse models of AML, displaying SAMHD1‐dependent synergy with ara‐C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara‐CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara‐C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.

Background There is a paucity of data on the prognostic value of programmed cell death protein 1 (PD-1) protein and gene expression in early breast cancer (BC) and the present study’s aim was to comprehensively investigate it.Methods The study consisted of three parts: a correlative analysis of PD-1 protein and gene expression from an original patient cohort of 564 patients with early BC; a systematic review and trial-level meta-analysis on the association between PD-1 protein expression and disease-free survival/overall survival (OS) in early BC; and a pooled gene expression analysis from publicly available transcriptomic datasets regarding PDCD1 expression.Results In the study cohort, PD-1 protein, but not gene expression, was associated with improved OS (HRadj=0.73, 95% CI 0.55 to 0.97, p=0.027 and HRadj=0.88, 95% CI 0.68 to 1.13, p=0.312, respectively). In the trial-level meta-analysis, PD-1 protein expression was not found to be statistically significantly associated with outcomes in the overall population. Finally, in the pooled gene expression analysis, higher PDCD1 expression was associated with better OS in multivariable analysis in the entire population (HRadj=0.89, 95% CI 0.80 to 0.99, p=0.025) and in basal-like tumours.Conclusions PD-1 protein and gene expression seem to be promising prognostic factors in early BC. Standardisation of detection and assessment methods is of utmost importance.

Abstract: Mutations of signal-induced proliferation-associated gene 1 (SIPA1), a RAP1 GTPase-activating protein, were reported in patients with juvenile myelomonocytic leukemia, a childhood myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Sipa1 deficiency in mice leads to the development of age-dependent MPN. However, Sipa1 expression in bone marrow (BM) microenvironment and its effect on the pathogenesis of MPN remain unclear. We here report that Sipa1 is expressed in human and mouse BM stromal cells and downregulated in these cells from patients with MPN or MDS/MPN at diagnosis. By using the Sipa1−/− MPN mouse model, we find that Sipa1 deletion causes phenotypic and functional alterations of BM mesenchymal stem and progenitor cells prior to the initiation of the MPN. Importantly, the altered Sipa1−/− BM niche is required for the development of MDS/MPN following transplantation of normal hematopoietic cells. RNA sequencing reveals an enhanced inflammatory cytokine signaling and dysregulated Dicer1, Kitl, Angptl1, Cxcl12, and Thpo in the Sipa1−/− BM cellular niches. Our data suggest that Sipa1 expression in the BM niche is critical for maintaining BM niche homeostasis. Moreover, Sipa1 loss–induced BM niche alterations likely enable evolution of clonal hematopoiesis to the hematological malignancies. Therefore, restoring Sipa1 expression or modulating the altered signaling pathways involved might offer therapeutic potential for MPN.

Austrian and German intellectuals were often disappointed when visiting contemporary Greece. This article examines various aspects of this tension between expectation and disappointment, as recorded in travel literature dating from the mid-nineteenth to the early twentieth century. It focuses on the nostalgia for Ancient Greece and its disillusionment due to the immense temporal distance. It also examines the portrayal of the landscape as a guarantee of continuity, in order to overcome this disappointment. To this Ancient-centered nostalgia this article juxtaposes one of a different kind, this time related to the Byzantine tradition. This nostalgia can be found in the German and Austrian travel literature that describes visits to Greek monasteries, especially on the Athos peninsula, and reaches its peak in the mystical overtones of travellers’ accounts in the twentieth century.