Patients with multiple myeloma (MM) carrying standard- or high-risk cytogenetic abnormalities (CAs) achieve similar complete response (CR) rates, but the later have inferior progression-free survival ...(PFS). This questions the legitimacy of CR as a treatment endpoint and represents a biological conundrum regarding the nature of tumor reservoirs that persist after therapy in high-risk MM. We used next-generation flow (NGF) cytometry to evaluate measurable residual disease (MRD) in MM patients with standard- vs high-risk CAs (n = 300 and 90, respectively) enrolled in the PETHEMA/GEM2012MENOS65 trial, and to identify mechanisms that determine MRD resistance in both patient subgroups (n = 40). The 36-month PFS rates were higher than 90% in patients with standard- or high-risk CAs achieving undetectable MRD. Persistent MRD resulted in a median PFS of ∼3 and 2 years in patients with standard- and high-risk CAs, respectively. Further use of NGF to isolate MRD, followed by whole-exome sequencing of paired diagnostic and MRD tumor cells, revealed greater clonal selection in patients with standard-risk CAs, higher genomic instability with acquisition of new mutations in high-risk MM, and no unifying genetic event driving MRD resistance. Conversely, RNA sequencing of diagnostic and MRD tumor cells uncovered the selection of MRD clones with singular transcriptional programs and reactive oxygen species–mediated MRD resistance in high-risk MM. Our study supports undetectable MRD as a treatment endpoint for patients with MM who have high-risk CAs and proposes characterizing MRD clones to understand and overcome MRD resistance. This trial is registered at www.clinicaltrials.gov as #NCT01916252.
•Achieving undetectable MRD overcomes the dismal prognosis of transplant-eligible multiple myeloma patients with high risk cytogenetics.•Characterization of MRD cells reveals greater clonal selection in standard-risk MM and ROS-mediated drug resistance in high-risk MM.
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Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, ...Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.
Abstract
Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and ...failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (
n
= 4) and patients with precursor (
n
= 8) and full-blown MM (
n
= 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27
−
and CD27
+
T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.
Background & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive biliary cystogenesis. Current therapies show short-term and/or modest beneficial effects. Cystic ...cholangiocytes hyperproliferate as a consequence of diminished intracellular calcium levels (Ca2+ i ). Here, the therapeutic value of ursodeoxycholic acid (UDCA) was investigated. Methods Effect of UDCA was examined in vitro and in polycystic (PCK) rats. Hepatic cystogenesis and fibrosis, and the bile acid (BA) content were evaluated from the liver, bile, serum, and kidneys by HPLC-MS/MS. Results Chronic treatment of PCK rats with UDCA inhibits hepatic cystogenesis and fibrosis, and improves their motor behaviour. As compared to wild-type animals, PCK rats show increased BA concentration (BA) in liver, similar hepatic Cyp7a1 mRNA levels, and diminished BA in bile. Likewise, BA is increased in cystic fluid of PLD patients compared to their matched serum levels. In PCK rats, UDCA decreases the intrahepatic accumulation of cytotoxic BA, normalizes their diminished BA in bile, increases the BA secretion in bile and diminishes the increased BA in kidneys. In vitro , UDCA inhibits the hyperproliferation of polycystic human cholangiocytes via a PI3K/AKT/MEK/ERK1/2-dependent mechanism without affecting apoptosis. Finally, the presence of glycodeoxycholic acid promotes the proliferation of polycystic human cholangiocytes, which is inhibited by both UDCA and tauro-UDCA. Conclusions UDCA was able to halt the liver disease of a rat model of PLD through inhibiting cystic cholangiocyte hyperproliferation and decreasing the levels of cytotoxic BA species in the liver, which suggests the use of UDCA as a potential therapeutic tool for PLD patients.
Information on the immunopathobiology of coronavirus disease 2019 (COVID-19) is rapidly increasing; however, there remains a need to identify immune features predictive of fatal outcome. This ...large-scale study characterized immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection using multidimensional flow cytometry, with the aim of identifying high-risk immune biomarkers. Holistic and unbiased analyses of 17 immune cell-types were conducted on 1,075 peripheral blood samples obtained from 868 COVID-19 patients and on samples from 24 patients presenting with non-SARS-CoV-2 infections and 36 healthy donors. Immune profiles of COVID-19 patients were significantly different from those of age-matched healthy donors but generally similar to those of patients with non-SARS-CoV-2 infections. Unsupervised clustering analysis revealed three immunotypes during SARS-CoV-2 infection; immunotype 1 (14% of patients) was characterized by significantly lower percentages of all immune cell-types except neutrophils and circulating plasma cells, and was significantly associated with severe disease. Reduced B-cell percentage was most strongly associated with risk of death. On multivariate analysis incorporating age and comorbidities, B-cell and non-classical monocyte percentages were independent prognostic factors for survival in training (n=513) and validation (n=355) cohorts. Therefore, reduced percentages of B-cells and non-classical monocytes are high-risk immune biomarkers for risk-stratification of COVID-19 patients.
BACKGROUNG: Multiparameter flow cytometry (MFC) is traditionally used to asses MRD in AML. However, its sensitivity and specificity lag behind other methods and this precludes a broader application ...in routine practice. It could be hypothesized the emerging multiomics such as single-cell RNA and antibody sequencing could uncover new differences from normal and leukemia associated immunophenotypes (DfN/LAIP). In addition, the identification of DfN/LAIP may be easier in PB when compared to the BM due to the scarcity of CD34+ HPC in the former sample. AIM: Uncover new DfN/LAIP for improved MFC discrimination between normal CD34+ HPC and blasts in the BM and PB of AML patients. METHODS: A total of 47 samples were analyzed in this study. Namely, paired BM and PB from 5 young healthy adults (20-23yo) and 5 PB samples from older healthy adults (47-88yo); paired BM and PB of 11 newly diagnosed AML (44-79yo) patients, and paired BM and PB collected in 5 of the 11 AML patients at the time of MRD assessment. We used fluorescence activated cell sorting (FACS) to isolate CD34+ HPC from the BM and PB of healthy adults as well as blasts from AML patients. scCITE-Seq with a panel of 600 genes and 50 oligo-conjugated antibodies, selected according to their role in stem cell identification, lineage differentiation, clonal hematopoiesis and aberrant expression in myeloid neoplasms was performed. After quality control filtering, a total of 111,334 cells were analyzed and differential expression of genes and antigens was considered if an adjusted P-value <.05 and a log 2FoldChange (FC) >|1| were observed. RESULTS: We first analyzed the phenotype of paired BM and PB 32,637 CD34+ HPC from younger healthy adults. There were 82 deregulated genes, 50 overexpressed in BM and 32 in PB. CLEC12A, CEBPA and HBB were amongst the top overexpressed genes in BM, whereas CRHBP, ADGRG6 and MEIS1 showed the highest upregulation in PB. These results suggest partial transcriptional overlap between BM and PB CD34+ HPC, with the former showing reduced differentiation. There were no differences in antigen expression. Next, we analyzed the potential effect of aging in the phenotype of 34,102 CD34+ HPC from the PB of young and older healthy adults. There were 34 genes differentially expressed including BCORL1, CBLB, HRAS and KDM6A. These findings might reflect an upregulation of genes associated with clonal hematopoiesis. Furthermore, there were 13 antigens differentially expressed including CD9, CD10, CD33, CD38, CD62L and CD371. The comparison of 56,387 blasts in paired BM and PB samples from newly diagnosed AML patients showed near overlap with only HBB being differentially expressed. There were no differences in antigen expression. We then investigated the extent of phenotypic differences between normal CD34+ HPC vs blasts in the BM and PB of healthy adult's vs AML patients. There were 224 and 15 differentially expressed genes between BM cell types, whereas in PB the numbers were 182 and 11, respectively. The markers that contributed the most in the discrimination between CD34+ HPC vs blasts in PB were WT1, ADM, HMOX1, DDIT3, HOPX, CD371, CD45RA, CD133 and CD47. The overlap between DfN/LAIP identified in PB and BM was 31%. Four of the five patients monitored after treatment were MRD positive in BM using MFC. Of note, BM and PB CD34+ HPC isolated from the five AML patients (including one MRD negative in BM) clustered separately of normal BM and PB CD34+ HPC from healthy adults. There was near overlap between BM and PB cells from AML patients. The markers that contributed the most in the discrimination between PB CD34+ HPCfrom healthy adult's vs resistant cells from AML patients were CXCL16, LILRA5, MME, CD274, CTSL, MINPP1 and AHSP. CONCLUSIONS: Normal CD34+ HPC in PB showed partial overlap with the BM counterpart, with a few phenotypic differences probably related to less differentiation and the effects of aging and clonal hematopoiesis. The phenotype of BM and PB blasts from newly diagnosed AML patients nearly overlapped, which indicates that DfN/LAIP may be equally applicable in BM and PB. Accordingly, the markers included in the scRNA/Ab-seq panel effectively discriminated CD34+ HPC from blasts regardless of the sample of origin. Furthermore, we showed phenotypic divergence between PB CD34+ HPC of healthy adults' vs AML patients after treatment. Taken together, this study identified new DfN/LAIP markers for improved assessment of MRD using MFC in the PB of AML patients.
Introduction Follicular lymphomas (FL) and diffuse large B cell lymphomas (DLBCL) share a common germinal center (GC) B cell precursor. Nevertheless, these lymphomas appear to originate from ...different developmental stages, through distinct pathogenetic mechanisms and their clinical behavior varies significantly, even among FL or DLBCL patients. To understand the molecular and transcriptional heterogeneity underlying this clinical diversity we conducted a multi-omic study in GC B cell lymphomas. Methods Our study comprised 3 DLBCL patients (1 non-GCB (germinal center B), 1 non-GCB CD5+, and 1 GCB) and 2 FL (1 with no transformation (ntFL) and 1 patient with transformation (tFL)) at diagnosis. We performed single-cell multiome-sequencing (scDNA-seq + scProtein) ( Tapestri platform -Mission Bio-) and single cell RNA-sequencing (scRNA-seq) ( Chromium system -10xGenomics-) from cell suspensions and spatial transcriptomics ( Visium technology -10xGenomics-) from paraffin blocks. Results Genotyping data were available for 19,700 cells. A total of 311 variants across the 5 samples passed all quality controls. We selected probably pathogenic and pathogenic variants for downstream analysis. Germline variants were discarded. In 3 of 5 patients, we detected 1 somatic and nonsynonymous variant defining the first clone (variants in KMT2D and NOTCH2 mutated in 6-38% of the cells) and another somatic and nonsynonymous variant, defining a subclone (variants in KMT2D, ATM, EZH2 mutated in 1-15% of the cells). In 1 of 5 patients (ntFL), we detected up to 4 mutations, acquired linearly ( B2M, GNA13, EZH2, RHOA mutated clone represents 8% of the cells). Remarkably, all patients showed the same pathogenic variant in TET2 (1-2% of the cells), as an independent clone. The highest molecular similarity was observed between the GCB DLBCL patient and the tFL patient. Both had the same variant in KMT2D as the first hit (later GCB-DLBCL acquired ATM, while tFL acquired EZH2 as a second hit). ATM and EZH2 mutations have been described in “bulk” studies conducted on DLBCL and FL patients, respectively. Regarding protein expression, there was a good correlation between GCB and non-GCB phenotypes studied by immunohistochemistry and scProtein (the expression of CD10 by proteomics was lower in non-GCB patients). TET2 clones described by scDNA-seq were enriched in CD5+ and CD19- cells, suggesting they belong to the T-lymphoid lineage. The other clones and subclones were enriched in CD19+ and CD5- cells ( Figure 1). Data from 54,024 single cells were obtained for scRNA-seq analysis, with an average of 10,805 cells (7,335-18,205) for each sample. Cell type annotation was performed based on the expression of canonical markers. We identified 13 different clusters, the main ones being: GC B cells, post-GC B cells, naïve B cells, myeloid cells, CD8+ cytotoxic T cells, CD4+ naïve T cells, CD4+ reg T cells, CD4+ helper T cells. All samples contained subpopulations of both GC and post-GC B cell clusters. However, GCB DLBCL and FL patients were significantly enriched in GC B cells (63.2 vs 22.2%; p<0.01), whereas non-GCB patients were significantly enriched in post-GC B cells (35.5 vs 2.9%; p<0.01). Part of the normal B-cell transcriptional function was preserved in all samples ( Figure 2). The analysis of the infiltrating T cells revealed that the CD4+/CD8+ T cells ratio progressively increased from ntFL (1.14) to tFL (2.25) and DLBCL (2.58). Furthermore, we estimated the exhaustion status of T cells by the expression of these five markers ( TIGIT, LAG3, CTLA4, HAVCR2, PDCD1). Exhaustion T cell markers were highly expressed in CD8+ cytotoxic T cells and cycling T cells and lower expressed in CD4+ reg T cells. Both DLBCL and tFL have a stronger pattern of exhaustion than ntFL. Finally, by the spatial transcriptomics analysis, we have identified distinct cell type proportions according to their matched single-cell RNA data. Conclusions Our results confirm the intra and intertumor heterogeneity in GC B cell lymphomas. Notably, the greatest molecular and transcriptional similarities are observed between the GCB DLBCL and tFL patients at diagnosis. Moreover, our findings support the critical role of the tumor microenvironment for the persistence and development of the tumor clone. The finding of a TET2 mutated clone in the infiltrating T cells of all the patients is particularly noteworthy, as it may suggest the possible presence of clonal lymphopoiesis.
Cl−/HCO 3− anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin‐stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood ...mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2‐deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR‐506) — predicted as candidate to target AE2 mRNA — for the decreased expression of AE2 in PBC. Real‐time quantitative polymerase chain reaction showed that miR‐506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR‐506 is up‐regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor‐mediated overexpression of miR‐506 in SV40‐immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR‐506 overexpression in three‐dimensional (3D)‐cultured H69 cholangiocytes blocked the secretin‐stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site‐directed mutagenesis demonstrated that miR‐506 specifically may bind the 3′untranslated region (3′UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR‐506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti‐miR‐506 was able to improve their AE2 activity. Conclusion: miR‐506 is up‐regulated in cholangiocytes from PBC patients, binds the 3′UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR‐506 may constitute a potential therapeutic target for this disease. (HEPATOLOGY 2012)
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