The Application of Nanotechnology for Quantification of Circulating Tumour DNA in Liquid Biopsies: A Systematic Review.

Technologies for quantifying circulating tumour DNA (ctDNA) in liquid biopsies could enable real-time measurements of cancer progression, profoundly impacting patient care. Sequencing methods can be too complex and time-consuming for regular point-of-care monitoring, but nanotechnology offers an alternative, harnessing the unique properties of objects tens to hundreds of nanometres in size. This systematic review was performed to identify all examples of nanotechnology-based ctDNA detection and assess their potential for clinical use. Google Scholar, PubMed, Web of Science, Google Patents, Espacenet and Embase/MEDLINE were searched up to 23rd March 2021. The review identified nanotechnology-based methods for ctDNA detection for which quantitative measures (e.g., limit of detection, LOD) were reported and biologically relevant samples were used. The pre-defined inclusion criteria were met by 66 records. LODs ranged from 10 zM to 50nM. 25 records presented an LOD of 10fM or below. Nanotechnology-based approaches could provide the basis for the next wave of advances in ctDNA diagnostics, enabling analysis at the point-of-care, but none are currently used clinically. Further work is needed in development and validation; trade-offs are expected between different performance measures e.g., number of sequences detected and time to result.

ATP-degrading ENPP1 is required for survival (or persistence) of long-lived plasma cells.

Survival of antibody-secreting plasma cells (PCs) is vital for sustained antibody production. However, it remains poorly understood how long-lived PCs (LLPCs) are generated and maintained. Here we report that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is preferentially upregulated in bone marrow LLPCs compared with their splenic short-lived counterparts (SLPCs). We studied ENPP1-deficient mice (Enpp1 -/- ) to determine how the enzyme affects PC biology. Although Enpp1 -/- mice generated normal levels of germinal center B cells and plasmablasts in periphery, they produced significantly reduced numbers of LLPCs following immunization with T-dependent antigens or infection with plasmodium C. chabaudi. Bone marrow chimeric mice showed B cell intrinsic effect of ENPP1 selectively on generation of bone marrow as well as splenic LLPCs. Moreover, Enpp1 -/- PCs took up less glucose and had lower levels of glycolysis than those of wild-type controls. Thus, ENPP1 deficiency confers an energetic disadvantage to PCs for long-term survival and antibody production.

Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1(lo) and B-1a.PC1(hi), differ significantly in IgH chain utilization. Adoptively transferred PC1(lo) cells secreted significantly more circulating natural IgM and intestinal IgA than PC1(hi) cells. In contrast, PC1(hi) cells produced more IL-10 than PC1(lo) cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1(hi) cells were also more efficient than PC1(lo) cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1(lo) cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1(hi) cells do not. We found that PC1(lo) cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1(hi) cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1(lo) and B-1a.PC1(hi) cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.

Characterization of monoclonal antibodies to the plasma cell alloantigen ENPP1.

The ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has documented roles in mineralization, nucleotide recycling, and insulin resistance. While ENPP1 was first identified as an alloantigen on mouse plasma cells (PCs), later studies revealed expression in many tissues. Previously described monoclonal antibodies against ENPP1 expressed at the cell surface recognized cells only from mice bearing the a allotype, ENPP1(a), precluding studies of mice bearing the alternative allele, ENPP1(b). Here, we characterize a novel anti-ENPP1 monoclonal antibody that recognizes both alleles and can be used for flow cytometry.

Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells.

Systemic antigen-induced PCs are generated in inductive lymphoid tissues. Some of them are selected to travel through the circulation and finally, to home onto BM niches. BM PCs show prolonged survival and secrete high-affinity antibodies. In this study, human PCs were isolated from tonsil, blood, and BM, their IGHV3 and IGHV6 genes were sequenced, and their SHM were evaluated. The SHM analysis reveals the existence of a maturational gradient in these genes, as demonstrated by a progressive increase in the frequency of total and R mutations and total and NC aa changes following the direction: tonsil --> blood --> BM. The ratio of R to S mutations in the CDR1 and -2, but not in the FRs, increases from tonsil to blood and BM; this parameter reaches a maximum threshold when more than 10 mutations/sequence occur. Further analyses indicate that CDR1 and CDR2 SHM followed different strategies to provide appropriate amino acid changes, but both exhibited maximal resistance to incorporating drastic molecular alterations in the BM PCs. Finally, all of the findings are similar in IGHV3 and IGHV6 sequences, indicating that they reflect general rules imposed by in vivo antigen selection.

IL-17 signaling-independent central nervous system autoimmunity is negatively regulated by TGF-beta.

Recent studies have established an important role of Th17 in induction of autoimmune diseases. We have found that although IL-17 receptor A (IL-17RA)(-/-) mice were resistant to experimental autoimmune encephalomyelitis, a small number of them developed milder clinical signs of this autoimmune disease. In addition, blockade of TGF-beta in IL-17RA(-/-) mice resulted in much more severe clinical signs of experimental autoimmune encephalomyelitis and significantly increased parenchymal lymphocyte infiltration in the CNS. Furthermore, the number of autoreactive Th1 cells was greatly increased in the inflamed spinal cord of IL-17RA(-/-) mice. These data support a role of IL-17RA-independent mechanisms in causing autoimmunity and its regulation by TGF-beta.

Increased survival is a selective feature of human circulating antigen-induced plasma cells synthesizing high-affinity antibodies.

The present study shows that tetanus toxoid (tet) booster releases to the human circulation 2 subsets of specific plasma cells (PCs), as defined by phenotype and morphology, which clearly differed in the staining capacity of their cytoplasmic antibodies (Abs) with fluorescein isothiocyanate (FITC)-labeled tet-fragment C (tetC). These cells, called tetCHIGH and tetCINT PCs according to their either high or intermediate FITC-tetC staining capacity, exhibit similar rapid temporary kinetics in the blood (5-8 days after boost), contain many cycling cells, express equivalent amounts of BLIMP-1 mRNA, and produce similar quantities of IgG. However, Abs synthesized by tetCHIGH PCs show a tetC affinity more than 10 times higher than that exhibited by tetCINT PC Abs, and indicated by IGVH sequence analysis. Chemotaxis to CXCL12, a requisite for bone marrow (BM) PC homing, is similar for both cell types. Circulating nonspecific and tetCINT PCs, but not tetCHIGH PCs, tend to undergo spontaneous apoptosis, as demonstrated by APO2.7 and activated caspase-3 expression, and cell recovery. These results indicate that tet booster generates 2 discrete subsets of specific PCs exhibiting different ranges of Ab affinity for the immunogen, and that only those synthesizing high-affinity Abs show enhanced survival. This inherent property may be essential for determining the BM fate of PCs secreting high-affinity Ab.

Higher maturity and connective tissue association distinguish resident from recently generated human tonsil plasma cells.

Human plasma cells (PC) are present in cell suspensions obtained from the tonsil by mechanical disaggregation (PC(MECH)). The present study shows that a collagenase treatment of tonsillar debris remaining after mechanical disaggregation yielded similar proportions of PC (PC(COLL)). Moreover, PC(MECH) were present in suspensions highly enriched in germinal center cells whereas PC(COLL) contained most of the IgA-secreting cells, suggesting their predominant location in follicular and parafollicular areas and connective tissue-rich zones such as tonsil subepithelium, respectively. Tonsil PC(MECH) and PC(COLL) shared the phenotype CD38(high) CD19(+) CD20(low) CD45(high), expressed equivalent amounts of PRDI BF1/Blimp-1 transcription factor, and carried similarly mutated IgVH6 genes. However, they differed in several features. 1) PC(MECH) still expressed the early B cell transcription factor BSAP and were HLA-DR(high); in contrast, PC(COLL) were BSAP(-)and HLA-DR(low). 2) PC(MECH) were CD95(+) and Bcl-2(+/-) whereas PC(COLL) showed CD95(+/-) and Bcl-2(+) expression; in addition, PC(MECH) exhibited increased spontaneous apoptosis. 3) The two PC subsets exhibited distinctive adhesion molecule profiles, since PC(COLL) expressed higher levels of CD31, CD44, and CD49d, but a lower level of CD11a than PC(MECH). These results suggest that PC(MECH) are recently generated, short-living PC, and PC(COLL) constitutes a subset with higher maturity and survival, which resides in connective tissue-rich areas.

The expression of PRDI-BF1 beta isoform in multiple myeloma plasma cells.

The PRDM1 gene, a master regulator of plasma cells (PC), can generate two transcription factor isoforms: PRDI-BF1alpha and PRDI-BF1beta. The present study shows that purified human normal PC have a significantly lower levels of PRDI-BF1beta expression than that in tumoral PC isolated from multiple myeloma (MM) (0.06+/-0.01 and 0.25+/-0.05, respectively; p<0.001). The role of this finding in MM is discussed.

Inorganic polyphosphate and specific induction of apoptosis in human plasma cells.

BACKGROUND AND OBJECTIVES: Inorganic polyphosphate (polyP), a ubiquitous phosphate polymer with ATP-like bonds, has recently been related to a variety of functions including blood coagulation and cell proliferation. We investigated the effects of polyP in the biology of human plasma cells (PC), responsible for the production and maintenance of antibodies in response to antigens. DESIGN AND METHODS: The U266 myeloma cell line was used to study whether polyP affects immunoglobulin (Ig) secretion and survival. Different human cell lines were used to test the specificity of polyP on viability. We analyzed Ig secretion of PC from bone marrow and peripheral blood after polyP addition. A conventional tetanus toxoid booster immunization was used to increase the proportion of PC in order to examine the ex vivo effects of polyP. We also tested the effects of polyP on primary myeloma cells. Ig secretion and apoptosis were determined by ELISA and FACS respectively. RESULTS: Addition of polyP to human PC produced an unexpected inhibition of Ig secretion and stimulation of apoptosis. PolyP generated apoptosis specifically in PC, myeloma (malignant PC) cell lines, primary myeloma cells, and B lymphoid cell lines. Normal B cells, T cells, total blood mononuclear cells, and non-lymphoid cell lines were not affected by polyP. In the U266 myeloma cell line, polyP induced externalization of phosphatidylserine, activation of caspase-3, and arrest of the cell cycle. The protective effects of interleukin-6 did not overcome the polyP-induced apoptosis Interpretation and CONCLUSIONS: Taken together, our results suggest for the first time the relevance of the use of polyP to the humoral immune response and open prospects for polyP as a novel therapy for myeloma.

Immunization-induced perturbation of human blood plasma cell pool: progressive maturation, IL-6 responsiveness, and high PRDI-BF1/BLIMP1 expression are critical distinctions between antigen-specific and nonspecific plasma cells.

The present study shows that reimmunization with tetanus toxoid (tet) caused a transient increase of the human blood plasma cell (PC) pool, detectable from 6th to 15th day postboost, as well as the temporal alteration of several PC features. Labeling of specific PC with FITC-tet C fragment (tetC) allowed kinetics analysis of the tetC(+) and tetC(-) PC, and revealed remarkable differences between them: 1) the kinetics of tetC(+) PC occurrence was exponential, and most of them appeared in a narrow time frame (5th to 8th day postboost), whereas the tetC(-) PC increase was lower (three to five times) and more prolonged (4th to 15th day postboost). 2) The tetC(+) PC subset contained a fraction of cycling cells, expressed high levels of DR, CD138, and CD126, and responded to IL-6 by improving their survival and Ig secretion; in contrast, the tetC(-) PC showed higher CXCR4 and lower DR and CD138, did not respond to IL-6, and contained a fraction of apoptotic cells. 3) Sequential phenotypic analysis revealed maturational changes within the tetC(+), but not tetC(-), PC subset; sequencing of tetC(+) PC IgVH genes showed clear features of Ag selection. 4) The tetC(+) PC expressed several times more positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1 transcription factor than the tetC(-) PC. 5) The tetC(-) PC and bone marrow resident PC similarly expressed low DR and high CXCR4, but differed in that the latter exhibited higher levels of CD31, CD138, and positive regulatory domain I- binding factor 1/B lymphocyte-induced maturation protein 1. These findings support the view that tetC(+) PC contain bone marrow PC precursors, and tetC(-) PC probably belong to a removable compartment of aged PC.

Identification of overexpressed genes in frequently gained/amplified chromosome regions in multiple myeloma.

BACKGROUND AND OBJECTIVES: Multiple myeloma (MM) is a malignancy characterized by clonal expansion of plasma cells. In 50% of the cases, the neoplastic transformation begins with a chromosomal translocation that juxtaposes the IGH gene locus to an oncogene. Gene copy number changes are also frequent in MM but less characterized than in other neoplasias. We aimed to characterize genes that are amplified and overexpressed in human myeloma cell lines (HMCL) to provide putative molecular targets for MM therapy. DESIGN AND METHODS: Nine HMCL were characterized by fluorescent in situ hybridization, comparative genomic hybridization (CGH) and cDNA microarrays for gene expression profiling and copy number changes. RESULTS: After defining the IGH-translocations present in the cell lines, we conducted expression-profiling analysis. Supervised analysis identified 166 genes with significantly different expression among the cell lines harboring MMSET/FGFR3 (4p16), MAF (16q) and CCND1 (11q13) rearrangements. Array-CGH was then performed. Five chromosomes recurrently affected by gains/amplifications in primary samples and cell lines were analyzed in detail. Sixty amplified and overexpressed genes were found and 25 (42%) of them were only overexpressed when amplified; moreover, six showed a significant association between overexpression and gain/amplification. We also found co-amplification and overexpression for genes located within the same amplicons, such as MALT1 and BCL2. INTERPRETATION AND CONCLUSIONS: Parallel analysis of gene copy numbers and expression levels by cDNA microarray in MM allowed efficient identification of genes whose expression levels are elevated because of increased copy number. This is the first time that MALT1 and BCL2 have been shown to be overexpressed and amplified in MM.

The heterogeneity shown by human plasma cells from tonsil, blood, and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression.

Plasma cells (PCs) are the final B-cell differentiation stage. Recent evidence reveals relevant functional differences within the PC compartment. In rodents, early PCs formed in secondary lymphoid tissues show enhanced apoptosis and short life span, whereas PCs present in a final destination organ, such as the bone marrow (BM), have reached a stable prolonged survival state. BM PCs arrive at this organ as a circulating precursor whose cellular nature remains uncertain. An initial aim of this study was to characterize this circulating cell. We hypothesized that antibody-secreting cells detectable in the human blood after immunization might be a candidate precursor. These cells were obtained from the blood of volunteers immunized 6 days earlier with tetanus toxoid (tet), and they were unambiguously identified as PCs, as demonstrated by their expression of the CD38(h) phenotype, by morphology, by immunoglobulin (Ig) intracytoplasmic staining, and by IgG-tet-secreting capacity in vitro. In addition, by using the common CD38(h) feature, human PCs from tonsil (as a possible source of early PCs), from blood from tet-immunized donors (as the putative precursors of BM PCs), and from BM (as a deposit organ) have been purified and their phenotypes compared. The results show that a variety of differentiation molecules, proteins involved in the control of apoptosis, the B-cell transcription factors, positive regulatory domain I-binding factor 1/B lymphocyte-induced maturation protein 1 and B cell-specific activating protein and, at least partially, the chemokine receptor CXCR4 were expressed by human PCs following a gradient of increasing maturity in the direction: tonsil-->blood-->BM. However, PCs from these different organs showed a local pattern of adhesion molecule expression. These observations are discussed in light of the complex physiology of the human PC compartment.