A comparative study of two routinely used protocols for ex vivo erythroid differentiation.

BACKGROUND: Erythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. RESULTS: In the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. CONCLUSIONS: We provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

Impact of body mass index on the prognosis of unresectable HCC patients receiving first-line Lenvatinib or atezolizumab plus bevacizumab.

INTRODUCTION: Overweight is a negative prognostic factor in the general population in the long term. However, the role of body mass index (BMI) in the short-mid term in advanced tumours is unclear. The present analysis investigates the role of BMI weight classes in a large sample of patients affected by HCC and receiving atezolizumab plus bevacizumab or lenvatinib as first-line treatment. METHODS AND MATERIAL: The cohort included consecutive patients affected by BCLC-c and BCLC-B HCC patients from a multicenter international study group who received atezolizumab plus bevacizumab or lenvatinib as first-line therapy. Population was stratified according to the BMI in under-, over- and normal-weight according to the conventional thresholds. The primary objective of the study was to evaluate the prognostic and predictive impact of BMI in patients affected by advanced or intermediate HCC. Survival curves were estimated using the product-limit method of Kaplan-Meier. The role of stratification factors was analysed with log-rank tests. RESULTS: 1292 consecutive patients with HCC were analysed. 466 (36%) patients were treated with lenvatinib and 826 (64%) patients were treated with atezolizumab plus bevacizumab. In the atezolizumab plus bevacizumab arm, 510 (62%) patients were normal-weight, 52 (6%) underweight and 264 (32%) overweight. At the univariate analysis for OS, underweight patients had significantly shorter OS compared to normal-weight patients, whereas no differences were found between normal-weight versus overweight. Multivariate analysis confirmed that underweight patients had significantly shorter OS compared to normal-weight patients (HR: 1.7; 95% CI: 1.0-2.8; p = .0323). In the lenvatinib arm, 26 patients (5.6%) were categorized as underweight, 256 (54.9%) as normal-weight, and 184 (39.5%) as overweight. At the univariate analysis for OS, no significant differences were found between normal-weight versus underweight and between normal-weight versus overweight, which was confirmed at multivariate analysis. CONCLUSION: Our analysis highlighted a prognostic role of BMI in a cohort of patients with advanced HCC who received atezolizumab plus bevacizumab, while no prognostic role for low BMI was apparent in patients who received lenvatinib.

Red Blood Cell Contribution to Thrombosis in Polycythemia Vera and Essential Thrombocythemia.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) characterized by clonal erythrocytosis and thrombocytosis, respectively. The main goal of therapy in PV and ET is to prevent thrombohemorrhagic complications. Despite a debated notion that red blood cells (RBCs) play a passive and minor role in thrombosis, there has been increasing evidence over the past decades that RBCs may play a biological and clinical role in PV and ET pathophysiology. This review summarizes the main mechanisms that suggest the involvement of PV and ET RBCs in thrombosis, including quantitative and qualitative RBC abnormalities reported in these pathologies. Among these abnormalities, we discuss increased RBC counts and hematocrit, that modulate blood rheology by increasing viscosity, as well as qualitative changes, such as deformability, aggregation, expression of adhesion proteins and phosphatidylserine and release of extracellular microvesicles. While the direct relationship between a high red cell count and thrombosis is well-known, the intrinsic defects of RBCs from PV and ET patients are new contributors that need to be investigated in depth in order to elucidate their role and pave the way for new therapeutical strategies.

Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification.

Locoregional Therapies and Remodeling of Tumor Microenvironment in Pancreatic Cancer.

Despite the advances made in treatment, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains dismal, even in the locoregional and locally advanced stages, with high relapse rates after surgery. PDAC exhibits a chemoresistant and immunosuppressive phenotype, and the tumor microenvironment (TME) surrounding cancer cells actively participates in creating a stromal barrier to chemotherapy and an immunosuppressive environment. Recently, there has been an increasing use of interventional radiology techniques for the treatment of PDAC, although they do not represent a standard of care and are not included in clinical guidelines. Local approaches such as radiation therapy, hyperthermia, microwave or radiofrequency ablation, irreversible electroporation and high-intensity focused ultrasound exert their action on the tumor tissue, altering the composition and structure of TME and potentially enhancing the action of chemotherapy. Moreover, their action can increase antigen release and presentation with T-cell activation and reduction tumor-induced immune suppression. This review summarizes the current evidence on locoregional therapies in PDAC and their effect on remodeling TME to make it more susceptible to the action of antitumor agents.

Circulating Tumor DNA in Gastric Adenocarcinoma: Future Clinical Applications and Perspectives.

Gastric cancer (GC) is still one of the most aggressive cancers with a few targetable alterations and a dismal prognosis. A liquid biopsy allows for identifying and analyzing the DNA released from tumor cells into the bloodstream. Compared to tissue-based biopsy, liquid biopsy is less invasive, requires fewer samples, and can be repeated over time in order to longitudinally monitor tumor burden and molecular changes. Circulating tumor DNA (ctDNA) has been recognized to have a prognostic role in all the disease stages of GC. The aim of this article is to review the current and future applications of ctDNA in gastric adenocarcinoma, in particular, with respect to early diagnosis, the detection of minimal residual disease (MRD) following curative surgery, and in the advanced disease setting for treatment decision choice and therapeutic monitoring. Although liquid biopsies have shown potentiality, pre-analytical and analytical steps must be standardized and validated to ensure the reproducibility and standardization of the procedures and data analysis methods. Further research is needed to allow the use of liquid biopsy in everyday clinical practice.

GRASP55 Is Dispensable for Normal Hematopoiesis but Necessary for Myc-Dependent Leukemic Growth.

Grasp55 is a ubiquitous Golgi stacking protein involved in autophagy, protein trafficking, and glucose deprivation sensing. The function of Grasp55 in protein trafficking has been attributed to its PDZ-mediated interaction with the C-terminal PDZ-binding motifs of protein cargos. We have recently shown that such an interaction occurs between Grasp55 and the adhesion molecule Jam-C, which plays a central role in stemness maintenance of hematopoietic and spermatogenic cells. Accordingly, we have found that Grasp55-deficient mice suffer from spermatogenesis defects similar to Jam-C knockout mice. However, whether Grasp55 is involved in the maintenance of immunohematopoietic homeostasis through regulation of protein transport and Jam-C expression remains unknown. In this study, we show that Grasp55 deficiency does not affect hematopoietic stem cell differentiation, engraftment, or mobilization, which are known to depend on expression of Grasp55-dependent protein cargos. In contrast, using an Myc-dependent leukemic model addicted to autophagy, we show that knockdown of Grasp55 in leukemic cells reduces spleen and bone marrow tumor burden upon i.v. leukemic engraftment. This is not due to reduced homing of Grasp55-deficient cells to these organs but to increased spontaneous apoptosis of Grasp55-deficient leukemic cells correlated with increased sensitivity of the cells to glucose deprivation. These results show that Grasp55 plays a role in Myc-transformed hematopoietic cells but not in normal hematopoietic cells in vivo.

Junctional adhesion molecule C (JAM-C) dimerization aids cancer cell migration and metastasis.

Most cancer deaths result from metastasis, which is the dissemination of cells from a primary tumor to distant organs. Metastasis involves changes to molecules that are essential for tumor cell adhesion to the extracellular matrix and to endothelial cells. Junctional Adhesion Molecule C (JAM-C) localizes at intercellular junctions as homodimers or more affine heterodimers with JAM-B. We previously showed that the homodimerization site (E66) in JAM-C is also involved in JAM-B binding. Here we show that neoexpression of JAM-C in a JAM-C-negative carcinoma cell line induced loss of adhesive property and pro-metastatic capacities. We also identify two critical structural sites (E66 and K68) for JAM-C/JAM-B interaction by directed mutagenesis of JAM-C and studied their implication on tumor cell behavior. JAM-C mutants did not bind to JAM-B or localize correctly to junctions. Moreover, mutated JAM-C proteins increased adhesion and reduced proliferation and migration of lung carcinoma cell lines. Carcinoma cells expressing mutant JAM-C grew slower than with JAM-C WT and were not able to establish metastatic lung nodules in mice. Overall these data demonstrate that the dimerization sites E66-K68 of JAM-C affected cell adhesion, polarization and migration and are essential for tumor cell metastasis.

Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

Dok1 and Dok2 Proteins Regulate Cell Cycle in Hematopoietic Stem and Progenitor Cells.

Dok1 and Dok2 proteins play a crucial role in myeloid cell proliferation as demonstrated by Dok1 and Dok2 gene inactivation, which induces a myeloproliferative disease in aging mice. In this study, we show that Dok1/Dok2 deficiency affects myeloproliferation even at a young age. An increase in the cellularity of multipotent progenitors is observed in young Dok1/Dok2-deficient mice. This is associated with an increase in the cells undergoing cell cycle, which is restricted to myeloid committed progenitors. Furthermore, cellular stress triggered by 5-fluorouracil (5-FU) treatment potentiates the effects of the loss of Dok proteins on multipotent progenitor cell cycle. In addition, Dok1/Dok2 deficiency induces resistance to 5-FU-induced hematopoietic stem cell exhaustion. Taken together, these results demonstrate that Dok1 and Dok2 proteins are involved in the control of hematopoietic stem cell cycle regulation.

Ptk7-Deficient Mice Have Decreased Hematopoietic Stem Cell Pools as a Result of Deregulated Proliferation and Migration.

Hematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. At the top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features are controlled through key microenvironmental cues and regulatory pathways, such as Wnt signaling. We showed previously that PTK7, a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesis has remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, with the highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss of Ptk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlated with increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling novel and unexpected functions for planar cell polarity pathways in HSC fate.

Adhesion receptors involved in HSC and early-B cell interactions with bone marrow microenvironment.

Hematopoiesis takes place in the bone marrow of adult mammals and is the process by which blood cells are replenished every day throughout life. Differentiation of hematopoietic cells occurs in a stepwise manner through intermediates of differentiation that could be phenotypically identified. This has allowed establishing hematopoietic cell classification with hematopoietic stem cells (HSCs) at the top of the hierarchy. HSCs are mostly quiescent and serve as a reservoir for maintenance of lifelong hematopoiesis. Over recent years, it has become increasingly clear that HSC quiescence is not only due to intrinsic properties, but is also mediated by cognate interactions between HSCs and surrounding cells within micro-anatomical sites called "niches". This hematopoietic/stromal crosstalk model also applies to more mature progenitors such as B cell progenitors, which are thought to reside in distinct "niches". This prompted many research teams to search for specific molecular mechanisms supporting leuko-stromal crosstalk in the bone marrow and acting at specific stage of differentiation to regulate hematopoietic homeostasis. Here, we review recent data on adhesion mechanisms involved in HSCs and B cell progenitors interactions with surrounding bone marrow stromal cells.

Hydroxycarbamide decreases sickle reticulocyte adhesion to resting endothelium by inhibiting endothelial lutheran/basal cell adhesion molecule (Lu/BCAM) through phosphodiesterase 4A activation.

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4ß1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

JAK2V617F activates Lu/BCAM-mediated red cell adhesion in polycythemia vera through an EpoR-independent Rap1/Akt pathway.

Polycythemia vera (PV) is characterized by an increased RBC mass, spontaneous erythroid colony formation, and the JAK2V617F mutation. PV is associated with a high risk of mesenteric and cerebral thrombosis. PV RBC adhesion to endothelial laminin is increased and mediated by phosphorylated erythroid Lu/BCAM. In the present work, we investigated the mechanism responsible for Lu/BCAM phosphorylation in the presence of JAK2V617F using HEL and BaF3 cell lines as well as RBCs from patients with PV. High levels of Rap1-GTP were found in HEL and BaF3 cells expressing JAK2V617F compared with BaF3 cells with wild-type JAK2. This finding was associated with increased Akt activity, Lu/BCAM phosphorylation, and cell adhesion to laminin that were inhibited by the dominant-negative Rap1S17N or by the specific Rap1 inhibitor GGTI-298. Surprisingly, knocking-down EpoR in HEL cells did not alter Akt activity or cell adhesion to laminin. Our findings reveal a novel EpoR-independent Rap1/Akt signaling pathway that is activated by JAK2V617F in circulating PV RBCs and responsible for Lu/BCAM activation. This new characteristic of JAK2V617F could play a critical role in initiating abnormal interactions among circulating and endothelial cells in patients with PV.

Genetic deletion of JAM-C in preleukemic cells rewires leukemic stem cell gene expression program in AML.

ABSTRACT: The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether "niche anchoring" of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of "not reached" after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.

ctDNA as promising tool for the assessment of minimal residual disease (MRD) and the need of an adjuvant treatment in gastroesophageal adenocarcinoma.

Gastroesophageal adenocarcinoma is a challenging disease due to its poor prognosis and the presence of few therapeutic options. For these reasons, it is mandatory to identify the subgroup of patients who are at high risk for relapse after curative-intention surgery. In the last years, liquid biopsy has aroused great interest in cancer treatment for its feasibility and the possibility to capture tumor heterogeneity in a real-time way. In postoperative setting, the interest is directed to the identification of Minimal Residual Disease (MRD), defined as isolated or small cluster of cancer cells that residues after curative-intention surgery, and are undetectable by conventional radiological and clinical exams. This review wants to summarize current evidence on the use of liquid biopsy in gastroesophageal cancer, focusing on the detection of ctDNA in the postoperative setting and its potential role as a guide for treatment decision.

Negative hyperselection of elderly patients with RAS and BRAF wild-type metastatic colorectal cancer receiving initial panitumumab plus FOLFOX or 5-FU/LV.

BACKGROUND: Upfront anti-EGFR therapy represents the standard of care for patients with left-sided, MSS/pMMR, RAS and BRAF wild-type mCRC. Molecular 'hyperselection' may optimize EGFR inhibition by detecting additional resistance alterations. MATERIALS AND METHODS: We used comprehensive genomic profiling on archival samples of elderly patients enrolled in the PANDA trial to detect: HER2 amplification/mutations; MET amplification; NTRK/ROS1/ALK/RET rearrangements; PIK3CA exon 20 mutations; PTEN alterations; AKT1 mutations; MAP2K1 mutations. We defined 'Gene Altered' (GA) patients whose tumour harboured at least one alteration, and 'Hyperselected' (HS) those without. Survival and tumour response outcomes were correlated to hyperselection status alone or combined with primary tumour sidedness or treatment arm. RESULTS: Genomic alterations were detected in 41/147 patients (27.9%). PFS, OS and ORR were inferior in GA versus HS (median PFS: 7.6 versus 12.8 months, HR = 2.08, 95% CI: 1.43-3.03, p < 0.001; median OS: 20.0 versus 29.5 months, HR = 1.82, 95% CI:1.23-2.69, p = 0.002; ORR: 51% versus 71%; OR = 0.43, 95% CI: 0.21-0.91, p = 0.02). In the multivariable models, the impact of hyperselection on PFS and OS was confirmed. Lower ORR was observed with 5-FU/LV/panitumumab in GA (40% versus 62%), but not in HS (70% versus 72%). GA was associated with worse survival and response regardless of primary tumour sidedness, whereas in the HS subgroup, right-and left sided tumours had similar outcomes. CONCLUSIONS: Molecular hyperselection and comprehensive genomic profiling have a potential usefulness in elderly patients with RAS/BRAF wild-type, pMMR/MSS mCRC, eligible for upfront EGFR inhibition.

The oncological multidimensional prognostic index is a promising decision-making tool: A real-world analysis in older patients with metastatic colorectal cancer.

BACKGROUND: Approximately 50% of colorectal cancers occur in older patients. International societies recommend geriatric tools to optimise treatment of older patients. Comprehensive Geriatric Assessment (CGA) is a multidimensional assessment used to classify patients as fit, vulnerable, or frail. The CGA-based oncological multidimensional prognostic index (onco-MPI) also classifies patients as high-, intermediate-, or low-risk based on tumour characteristics. We investigated the role of CGA and onco-MPI in older patients with metastatic colorectal cancer (mCRC) in a real-world setting. METHODS: Data for consecutive mCRC patients aged ≥70 years were retrieved from a prospectively maintained database from 2010 to 2020. We analyzed patients' and tumours' characteristics, and the CGA domains. Onco-MPI was calculated by a validated algorithm derived from CGA domains. Pearson's test was used to verify whether onco-MPI scores and chemotherapy administration were correlated. RESULTS: The study included 488 mCRC patients with a mean age of 76.1 years. According to CGA, 52% of patients were fit, 28% vulnerable, and 20% frail. According to onco-MPI, 9% were low, 54% intermediate, and 37% high-risk. The median OS was 22.7 months. The following factors improved OS: 0-1 ECOG PS, low onco-MPI, fit based on CGA, chemotherapy administration, and doublet regimen. Chemotherapy administration significantly correlated with onco-MPI scores, leading to a survival gain regardless of the risk subgroups. First-line regimen had no impact on survival across the CGA and onco-MPI categories. CONCLUSION: CGA and onco-MPI scores confirmed their prognostic impact in older mCRC patients and may aid in decision-making and subgroup stratification in dedicated trials.

The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5.

Tissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center-experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.