NK cell dysfunction with down‐regulated CD16 and up‐regulated CD56 molecules in patients with esophageal squamous cell carcinoma

NK cells can be divided into two subsets, CD56^dim and CD56^bright NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56^dim or CD56^bright NK cells gated on CD56(+)CD3(–) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab‐mediated antibody‐dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)‐expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56^dim and CD56^bright NK cells between ESCC patients and healthy donors, down‐regulated CD16 and up‐regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab‐mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down‐regulated CD16 and up‐regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF‐beta1 partially contributed to down‐regulation of CD16 on NK cells. Down‐regulated CD16 and up‐regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.     

Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34⁺ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-α, TGF-β and Flt-3 ligand (fold increase of CD1a⁺ cells = 102 ± 32 after 2 weeks of culture). CD34⁺ cells were also grown under continuous flow conditions in an artificial capillary system; after 14 d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 ± 2 v 18.9 ± 4) but the percentage of CD1a⁺/CD83⁺/CD80⁺ cells was considerably higher, whereas the CD14⁺ cells were significantly reduced (8.9 ± 2 v 26 ± 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a⁺/S100⁺/CD83⁺/CD80⁺/CD14^?‘large cells’ with great internal complexity (mature DCs); the second including ‘small cells’ either CD33⁺/CD14⁺, CD33⁺/CD15⁺ or CD33⁺/CD13^±/CD14^?. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.     

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G-CSF)/chemotherapy mobilized peripheral blood CD34⁺ cells to selective erythroid differentiation in liquid culture with an average 28-fold increase in the total cell number after 21 d. From day 6 of culture, cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34⁺ cells were cultured with EPO plus SCF in serum-free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34⁺ cells underwent granulocytic/monocytic differentiation in liquid culture in response to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and SCF, demonstrating that these CD34⁺ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34⁺ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34⁺ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G-CSF/chemotherapy mobilized CD34⁺ progenitor cells in liquid culture. The absence of cytokines such as GM-CSF and IL-3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.     

Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective as with Dexa-BEAM plus G-CSF in lymphoma patients

We compared retrospectively the efficacy of granulocyte colony stimulating factor (G-CSF) alone with chemotherapy plus G-CSF in mobilizing CD34-positive cells in patients with malignant lymphoma. 35 patients underwent peripheral blood stem cell (PBSC) collection following mobilization either with 24 μg/kg G-CSF for 4 consecutive days (n = 18) or Dexa-BEAM chemotherapy plus 5 μg/kg G-CSF (n = 17). High-dose G-CSF was well tolerated with only slight bone pain and/or myalgia. The Dexa-BEAM therapy required hospitalization with a median duration of 21 d. The median number of apheresis procedures in both groups was two (range two to four), resulting in a median of 5.3 and 5.1 × 10⁶ CD34⁺ cells/kg. No patients in the G-CSF group, but one in the Dexa-BEAM group, failed to reach the target of collecting >2.0 × 10⁶ CD34⁺ cells/kg. The number of CFU-GM (10.4 v 6.0 × 10⁵/kg) and of BFU-E (10.6 v 4.5 × 10⁵/kg; P = 0.04) was higher in the G-CSF group than in the Dexa-BEAM group. A subset analysis of CD34⁺ cells was performed in 16 patients showing a higher mean of Thy-1 (CD90w) coexpression in the G-CSF than in the Dexa-BEAM group (4.8 v 1.8%, P = 0.12). Additionally the percentage of CD34⁺/CD38^? cells was higher in the G-CSF group (10.6% v 8.8%). However, these differences were not stastistically significant. The median time to leucocyte and platelet engraftment after high-dose chemotherapy was slightly shorter in the G-CSF than in the Dexa-BEAM group (9 v 10 and 12 v 13.5 d, respectively). These results demonstrate that high-dose G-CSF is as effective as Dexa-BEAM plus G-CSF in mobilizing peripheral blood stem cells and produces prompt engraftment. The major advantages of G-CSF mobilization were the safe outpatient self-application and the fixed-day apheresis.     

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors

Circulating CD34⁺ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34^? contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3⁺ T-lymphocytes (range 2–12%), CD3^?/CD16⁺/CD56⁺ natural killer cells (range 2–11%) and rare mature CD15⁺/CD11b⁺ granulocytes (range 1–2%). CD34⁺ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34⁺ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)₂D₃, parathyroid hormone, interleukin-11 and of other cytokines on osteoclast development in humans.     

Circulating CD20dim T-lymphocytes increase with age: evidence for a memory cytotoxic phenotype

Summary The CD20 antigen has been regarded as a B lineage specific, 35 kDa, non-glycosylated membrane phosphoprotein, which functions as either a Ca²⁺ ion channel or as a regulatory protein of such a channel. Weak expression of CD20 (CD20^dim), however, has recently been reported on a sub-population of T lymphocytes. We present results which confirm the existence of a CD20^dim T lymphocyte population and show that such cells have a reduced antibody-binding capacity, when compared to CD20^bright B-cells (10 337±642 and 346 311 ± 24 264 respectively). In addition, CD20^dim cell counts vary with age, with the highest levels occurring in octogenarians: cord blood 0.3 ± 0.1% (n = 13), 20–60 year-old group 2.1 ± 1.1% (n = 18) and individuals ≥ 61 years of age 6.9 ± 3.2% (n = 10) (P < 0.001). Further characterization of CD20^dim T cells, using three colour flow cytometry, demonstrated a predominantly memory cytotoxic phenotype, in that the cells were CD8⁺CD28⁺CD45RO⁺T-CRαβ⁺CD38^?HLA-DR^?.     

Generation of human natural killer cells from peripheral blood CD34+ cells mobilized by granulocyte colony-stimulating factor

We studied the generation of human natural killer (NK) cells from CD34⁺ cells that were isolated from peripheral blood stem cells (PBSC) mobilized by granulocyte colony-stimulating factor (G-CSF). The isolated CD34⁺ cells were cultured in the presence of a combination of interleukin-1 (IL-1α), IL-2, and stem cell factor for 5 weeks without marrow stroma. We found that the CD34⁺ cells isolated from G-CSF-mobilized PBSC (G-CSF/PBSC) could differentiate into a population of NK cells which were CD56^+(bright)/CD3^? and showed morphologic characteristics of large granular lymphocytes. Immunophenotypic analysis of the NK cells thus generated showed that a small proportion of them expressed CD2, CD8 and CD16 surface markers and approximately half of them coexpressed CD7. This NK population exhibited cytotoxic activity against a NK-sensitive cell line, K562. These observations suggest that CD34⁺ cells from G-CSF/PBSC contain precursors of NK cells that can differentiate into functional NK cells.     

Production of matrix metalloproteinase-9 by cord blood CD34<Superscript>+</Superscript> cells and its role in migration

The transmigration of hematopoietic progenitor cells is a crucial step in the homing of transplanted stem cells into bone marrow (BM) microenvironment; however, the molecular basis for this is not fully understood. Matrix metalloproteinases (MMPs), which are implicated in the migration of leukocytes, are important in degrading components of extracellular matrix molecules. In this study, using zymographic analysis and enzyme-linked immunosorbent assay (ELISA), we investigated the production of MMP-9 in CD34⁺ cells from cord blood (CB) and BM, compared their spontaneous migration across a reconstituted basement membrane-coated filter in transwell, and studied the role of MMP-9 in the transmigration. Zymography and ELISA showed that MMP-9 is produced by freshly isolated CD34⁺ stem/progenitor cells obtained from CB. CB CD34⁺ cells showed significantly higher migrational capacity than BM CD34⁺ cells (p=0.008). Furthermore, the migrational ability of CB CD34⁺ cells over the extracellular matrix (ECM) was significantly inhibited by the inhibitor of MMP, o-phenanthroline and anti-MMP-9 monoclonal antibody (73.3±11.8% and 37.5±10.4% inhibition, respectively). Our results strengthen the potential role of MMP-9 in the higher migrational capacity of CB CD34⁺ cells, which may be beneficial to homing of these cells to the BM environment.     

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein

Abstract: We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34⁺ blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte-colony stimulating factor (G–CSF) by peripheral blood (PB)-derived CD34⁺c-kit^? cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)-3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34⁺c-kit^high cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM–CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34⁺c-kit^{high or low} cells in the presence of Epo. Replating of E-Mix colonies derived from CD34⁺c-kit^high cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34⁺c-kit^low cells which represent a more immature population than CD34⁺c-kit^high cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.     

Establishment and characterization of a mantle cell lymphoma cell line

A mantle cell lymphoma (MCL) cell line (JeKo-1) was established from peripheral blood mononuclear cells of a patient with a large cell variant of MCL showing leukaemic conversion. JeKo-1 cells were Epstein-Barr virus negative and showed a B-cell phenotype with IgM⁺, IgD⁺, CD3^?, CD5⁺, CD10^?, CD19⁺, CD20⁺ and CD23^?; they overexpressed cyclin Dl, Bcl-2, c-Myc and Rb proteins. Bcl-1/J_H gene rearrangement was confirmed by polymerase chain reaction, although karyotypic analysis showed 40/41 chromosomes devoid of apparent t(11;14)(q13;q32) translocation. JeKo-1 cells were highly tumourigenic in SCID mice.     

Interferon therapy shifts natural killer subsets among Egyptian patients with chronic hepatitis C

Natural killer cells can be divided into five subpopulations based on the relative expression of CD16 and CD56 markers. The majority of natural killer cells are CD56^dim, which are considered to be the main cytotoxic effectors. A minority of the natural killer cells are CD56^bright, and function as an important source of immune-regulatory cytokines. Shifts of these subsets have been reported in patients with chronic hepatitis C virus infection. We sought to investigate the shift of natural killer subsets among Egyptian patients with chronic HCV and to analyze the influence of interferon therapy on this shift. We applied a flow cytometric analysis of peripheral blood natural killer subsets for 12 interferon-untreated and 12 interferon-treated patients with chronic HCV, in comparison to 10 control subjects. Among interferon-untreated patients, there was a significant reduction of CD56^-16⁺ (immature natural killer) cells. Among interferon-treated patients, the absolute count of natural killer cells was reduced, with expansion of the CD56^bright subset and reduction of the CD56^dim16⁺ subset. Natural killer subset counts were not significantly correlated to HCV viral load and were not significantly different among interferon responders and non-responders. In conclusion, HCV infection in Egyptian patients has been observed to be statistically and significantly associated with reduction of the CD56^-16⁺NK subset, while a statistically significant expansion of CD56^bright and reduction of CD56^dim16⁺ subsets were observed after interferon therapy. Further studies are required to delineate the molecular basis of interferon-induced shift of natural killer subsets among patients with HCV.     

Immunoadsorption of Immunoglobulins Alters Intracytoplasmic Type 1 and Type 2 T Cell Cytokine Production in Patients with Refractory Autoimmune Diseases

Abstract: Intracellular cytokine staining and flow cytometry were used to investigate whether immunoadsorption (IA) of immunoglobulins alters intracytoplasmic cytokine production in CD4⁺ and CD8⁺ T cells from the blood of patients with refractory rheumatoid arthritis (n = 7), membrane proliferative glomerulonephritis (n = 1), and Goodpasture's syndrome (n = 1). Four patients (Group 1) showed severely depressed production of TNF‐α, IL‐2, IFN‐γ, and IL‐4 by CD4⁺ and CD8⁺ T cells and responded to 3 IA sessions with significant increases in CD4⁺TNF‐α⁺, CD4⁺IL‐2⁺, and CD8⁺IL‐2⁺ T cells. Also, a tendency toward increased percentage levels of CD4⁺ T cells producing IFN‐γ or IL‐4 and of CD8⁺ T cells producing either TNF‐α or IFN‐γ was seen, but due to the small number of patients investigated, these differences did not attain statistic significance. Group 2 (n = 5) showed unimpaired intracellular cytokine levels and responded to IA with a heterogeneous pattern of changes in TNF‐α, IL‐2, IFN‐γ, and IL‐4 production, but these alterations were smaller than those in Group 1. The present findings indicate that the extracorporeal removal of immunoglobulins by anti‐IgG or protein A adsorber columns has an impact on T cell immunity and suggest that modulating effects on cellular immune system function are involved in the mode of action of IA.     

Expansion of circulating CD56bright natural killer cells in patients with JAK2‐positive chronic myeloproliferative neoplasms during treatment with interferon‐α

In recent years, major molecular remissions have been observed in patients with JAK2‐positive chronic myeloproliferative neoplasms (MPNs) after therapy with IFN‐α. IFN‐α is known to have altering effects on immune cells involved in immune surveillance and might consequently enhance anti‐tumor immune response against the JAK2‐mutated clone. The objective of this study was to investigate circulating levels and phenotype of natural killer cells in 29 JAK2‐positive MPN patients during IFN‐α treatment. Furthermore, functional studies of NK cells upon target‐cell recognition and cytokine stimulation were performed. The CD56^bright and CD56^dim NK cell subtypes display different properties in terms of cytokine production and cytotoxicity, respectively. Our results show a significant increase in the proportion of CD56^bright NK cells and a decreasing CD56^dim population during treatment with IFN‐α compared to patients that are untreated, treated with hydroxyurea and healthy controls, P < 0.0001. Furthermore, an overall increase in cytokine‐dependent (IL‐12 and IL‐15) IFN‐γ expression by CD56^dim NK cells during IFN‐α treatment was observed. In contrast, our data indicate a compromised NK cell response to target‐cell recognition during treatment with IFN‐α in four patients. We also report low levels of circulating NK cells in untreated patients compared to healthy donors, patients treated with hydroxyurea and IFN‐α, P = 0.02. Based on our findings, one might speculate whether treatment with IFN‐α skews the human NK population toward a helper type that may assist in CD8⁺ T cell priming in lymphoid tissues at the expense of their immediate cytotoxic functions in peripheral blood and tissues.     

Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib

Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45^neg/CD34^bright), HPCs (CD45^dim/CD34^bright), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50 mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34^bright HPCs and monocytes decreased during treatment, CD34^bright CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; P = 0.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34^bright cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessels. Laura Vroling and Astrid A. M. van der Veldt contributed equally to this study.     

Rheumatoid synovium is enriched in CD45RBdim mature memory T cells that are potent helpers for B cell differentiation

Objective. To delineate the phenotype and function of synovial T cells in rheumatoid arthritis (RA). Methods. T cells from normal subjects or from RA peripheral blood (PB), synovial fluid (SF), or synovial tissue (ST) were analyzed phenotypically and functionally. Results. RA SF and ST T cells were found to be markedly enriched in CD45RA^dim, CD45RO+, CD45RB^dim mature memory cells, whereas in the PB, CD45RA^bright naive T cells were more frequent than CD45RO+ memory T cells, and only a minority were CD45RB^dim. SF and ST T cells proliferated less well and produced less interleukin-2 in response to mitogenic stimuli than did PB T cells. However, synovial T cells effectively promoted the production of Ig from normal B cells. Moreover, PB and synovial T cells differed in their capacity to down-regulate immunoglobulin production. Anti-CD3—stimulated PB T cells suppressed Ig production unless their proliferation was prevented with mitomycin C. In contrast, synovial T cells were potent helpers of B cell Ig production regardless of antecedent treatment with mitomycin C. To examine the relationship between the CD45RB^dim phenotype and B cell help, CD45RB^dim T cells were sorted from PB. As opposed to the findings with synovial T cells, suppression by control PB CD45RB^dim T cells was observed, but only when large numbers were employed. B cell Ig production was enhanced after treatment of PB CD45RB^dim T cells with mitomycin C. In contrast, healthy control sorted CD45RB^bright or sorted CD4+, CD45RO+, CD45RB^bright T cells did not support Ig secretion. After treatment with mitomycin C, both of these populations were more effective helpers of Ig production. Conclusion. RA synovium is enriched in differentiated CD45RB^dim memory T cells with potent helper activity and diminished capacity to down-regulate B cells, strongly implying an active role for these cells in the production of Ig in the synovium, and thus in the propagation of disease.     

Haemopoietic defect and decreased expansion potential of bone marrow autografts from patients with acute myeloid leukaemia in first remission

Autologous bone marrow (BM) transplantation for acute myeloid leukaemia (AML) in complete remission (CR) is frequently followed by a slow haemopoietic recovery. We assessed the haemopoietic capacity of purified BM stem cell (CD34⁺DR^?) and progenitor cell (CD34⁺DR⁺) populations from patients with AML in CR, and compared these data with those of normal BM. The feasibility of ex vivo expansion in stroma-conditioned medium supplemented with cytokines was also investigated. The number of CFU-GM produced by initial patient CD34⁺DR^? cells was decreased compared to normal, whereas these values were similar to normal for CD34⁺DR⁺ cells. BFU-E, HPP-CFC and LTC-IC were reduced for both patient CD34⁺DR^? and CD34⁺DR⁺ subpopulations. In contrast to normal, the patient CD34⁺DR^? fraction was not enriched in LTC-IC. CFU-GM expansion from patient CD34⁺DR^? cells was poor and decreased after 14 d of culture. No HPP-CFC expansion could be observed for patient cells. LTC-IC were below the level of detection after 14–21 d of expansion culture of CD34⁺DR^? patient cells, whereas they were variably maintained or expanded for normal cells. After expansion culture, cytogenetic and/or FISH analyses did not reveal the anomalies present at diagnosis, regardless of the cell subpopulation analysed. In conclusion, BM cells of patients with AML in CR show a profound defect at the level of a stem cell enriched population. No meaningful ex vivo expansion could be obtained in culture conditions allowing for a significant expansion from a normal stem cell population.     

CD5/CD20 expression on circulating B cells in HCV-related chronic hepatitis and mixed cryoglobulinemia

The role of CD5⁺ B cells in patients with HCV infection and HCV-related disorders, including mixed cryoglobulinemia (MC), has been addressed in previous reports with conflicting results. We established a correlation between CD5/CD20 expression on circulating B lymphocytes, characterizing monoclonal B cell lymphocytosis (MBL), and clinical features in a cohort of 45 patients with chronic HCV hepatitis [without MC: 23 patients (MC- group); with MC: 22 patients (MC+ group)], and 45 HCV-negative healthy subjects as controls. By flow cytometry analysis, three B cells phenotypes were singled out: 1) CD5⁺CD20^dim (CLL-like phenotype); 2) CD5⁺CD20^bright (atypical phenotype); and 3) CD5^-CD20⁺ phenotype. CD5⁺CD20^bright cells were reduced in MC- patients (p=0.049). CD5⁺CD20^dim B cells were significantly higher in group B than in the control group (p=0.003). ROC curve analysis in MC+ patients showed the highest positive likelihood ratio at ≥7.35% (p=0.008) for CLL-like phenotype and at ≤63.6% (p=0.03) for the CD5^-CD20⁺ B cell phenotype. HCV infection was associated with a higher frequency of CLL-like (odds ratio=16, p=0.002) and a lower frequency of atypical (odds ratio: 3.1, p=0.02) and CD5^-CD20⁺ (odds ratio: 11, p=0.01) phenotypes. The association with higher levels of CLL-like phenotype progressively increased from group of MC- patients (odds ratio: 9.3, p=0.04) to the group of MC+ patients (odds ratio: 25.1, p=0.0003).ConclusionsThe occurrence of a CLL-like pattern may allow to identify HCV-infected patients at risk of developing MC and eventually non-Hodgkin lymphoma, who should require a closer surveillance and a longer follow-up.     

Unravelling the relevance of CLEC12A as a cancer stem cell marker in myelodysplastic syndrome

Evidence of distinct disease propagating stem cells in myelodysplastic syndrome (MDS) has emerged in recent years. However, immunophenotypic characterization of these cancer stem cells remains sparse. In acute myeloid leukaemia (AML), we have previously described aberrant expression of the C‐type lectin domain family 12, member A (CLEC12A) as a stable and reliable marker of leukaemia blasts and as a tool for assessing minimal residual disease. Furthermore, CLEC12A has been proposed as a promising marker of leukaemic stem cells in AML. The role of CLEC12A in MDS, however, remains to be elucidated. In this study, we found CLEC12A aberrantly expressed on the CD34⁺CD38^? cell compartment in 71% (22/31) of MDS patients, distributed across all Revised International Prognostic Scoring System risk groups. We showed that the CD34⁺CD38^?CLEC12A⁺ cells were indeed malignant and possessed functional stem cell properties in the long‐term colony‐initiating cell assay. As opposed to reported findings in AML, we showed that cancer stem cells from MDS samples derived from both CLEC12A positive and negative CD34⁺CD38^? subpopulations. Due to the absence of CLEC12A on normal haematopoietic stem cells, CLEC12A stem cell immunophenotyping may contribute to diagnosing and monitoring MDS patients and could furthermore add knowledge about disease propagating cells in MDS.