The profile and prognostic significance of bone marrow T-cell differentiation subsets in adult AML at diagnosis.

Background: T lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited. Patients and methods: Fresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry. Results: CD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) (P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4-22.2), P = 0.017 and 4.8 [1.3-17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1-9.5), P = 0.029; 4.0 (1.4-11.5), P = 0.010), respectively. Conclusions: AML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes.

Measurable residual disease testing by next generation sequencing is more accurate compared with multiparameter flow cytometry in adults with B-cell acute lymphoblastic leukemia.

Results of measurable residual disease (MRD)-testing by next-generation sequencing (NGS) correlate with relapse risk in adults with B-cell acute lymphoblastic leukemia (ALL) receiving chemotherapy or an allotransplant from a human leukocyte antigen (HLA)-identical relative or HLA-matched unrelated donor. We studied cumulative incidence of relapse (CIR) and survival prediction accuracy using a NGS-based MRD-assay targeting immunoglobulin genes after 2 courses of consolidation chemotherapy cycles in 93 adults with B-cell ALL most receiving HLA-haplotype-matched related transplants. Prediction accuracy was compared with MRD-testing using multi-parameter flow cytometry (MPFC). NGS-based MRD-testing detected residual leukemia in 28 of 65 subjects with a negative MPFC-based MRD-test. In Cox regression multi-variable analyses subjects with a positive NGS-based MRD-test had a higher 3-year CIR (Hazard Ratio [HR] = 3.37; 95 % Confidence Interval [CI], 1.34-8.5; P = 0.01) and worse survival (HR = 4.87 [1.53-15.53]; P = 0.007). Some data suggest a lower CIR and better survival in NGS-MRD-test-positive transplant recipients but allocation to transplant was not random. Our data indicate MRD-testing by NGS is more accurate compared with testing by MPFC in adults with B-cell ALL in predicting CIR and survival. (Registered in the Beijing Municipal Health Bureau Registration N 2007-1007 and in the Chinese Clinical Trial Registry [ChiCTR-OCH-10000940 and ChiCTROPC-14005546]).

A lasso and random forest model using flow cytometry data identifies primary myelofibrosis.

Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38- cells on CD34+cells, CD13dim+CD11b- cells in granulocytes, CD38str+CD19+/-plasma, and CD123+HLA-DR-basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38- on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.

Prognostic significance of the frequencies of bone marrow lymphocyte subsets in adult acute myeloid leukemia at diagnosis.

INTRODUCTION: Immune microenvironment plays an important role in the occurrence and development of acute myeloid leukemia (AML). Studies assessing the prognostic significance of bone marrow (BM) lymphocyte subsets' frequencies at diagnosis in patients with AML were limited. METHODS: Fresh BM samples collected from 97 adult AML patients at diagnosis were tested for lymphocyte, T, CD4+ T, CD8+ T, γδT, NK, and B cell frequencies using multi-parameter flow cytometry. RESULTS: Low frequencies of lymphocytes, T, CD4+ T, and CD8+ T cells were associated with significantly lower rates of one-course complete remission (CR) (all p < 0.05). Moreover, the frequency of CD4+ T cells independently predicted one-course CR achievement (p = 0.021). Low frequencies of T and CD8+ T cells were significantly associated with lower relapse-free survival (RFS) rates (p = 0.032; 0.034), respectively, and a low frequency of CD8+ T cells was associated with a significantly lower overall survival (OS) rate (p = 0.028). Combination of frequency of CD8+ T cells and ELN risk stratification showed that patients with ELN-intermediate/adverse risk + high CD8+ T cell frequency had a similar RFS rate to those with ELN-favorable risk + high CD8+ T cell frequency and those with ELN-favorable risk + low CD8+ T cell frequency (p = 0.88; 0.76), respectively. The RFS rate of patients with ELN intermediate/adverse risk + low CD8+ T cell frequency was significantly lower than that of all aforementioned patients (p = 0.021; 0.0007; 0.028), respectively. CONCLUSION: The frequencies of BM lymphocyte subsets at diagnosis predicted clinical outcomes and could help improve risk stratification in AML.

Colorectal cancer cell-secreted exosomal miRNA N-72 promotes tumor angiogenesis by targeting CLDN18.

Angiogenesis is essential for the growth and metastasis of several malignant tumors including colorectal cancer (CRC). The molecular mechanism underlying CRC angiogenesis has not been fully elucidated. Emerging evidence indicates that secreted microRNAs (miRNAs) may mediate the intercellular communication between tumor cells and neighboring endothelial cells to regulate tumor angiogenesis. In addition, exosomes have been shown to carry and deliver miRNAs to regulate angiogenesis. miRNA N-72 is a novel miRNA that plays a regulatory role in the EGF-induced migration of human amnion mesenchymal stem cells. However, the relation between miRNA N-72 and cancer remains unclear. We here found that CRC cells could secrete miRNA N-72. A high miRNA N-72 level was detected in the serum of CRC patients and the cultured CRC cells. Moreover, the CRC cell-secreted miRNA N-72 could promote the migration, tubulogenesis, and permeability of endothelial cells. In addition, the mouse xenograft model was used to verify the facilitating effects of miRNA N-72 on CRC growth, angiogenesis, and metastasis in vivo. Further mechanism analysis revealed that CRC cell-secreted miRNA N-72 could be delivered into endothelial cells via exosomes, which then inhibited cell junctions of endothelial cells by targeting CLDN18 and consequently promoted angiogenesis. Our findings reveal a novel mechanism of CRC angiogenesis and highlight the potential of secreted miRNA N-72 as a therapeutic target and a biomarker for CRC.

Identification of an optimized glycolytic-related risk signature for predicting the prognosis in breast cancer using integrated bioinformatic analysis.

Aberrant metabolic disorders and significant glycolytic alterations in tumor tissues and cells are hallmarks of breast cancer (BC) progression. This study aims to elucidate the key biomarkers and pathways mediating abnormal glycolysis in breast cancer using bioinformatics analysis. Differential genes expression analysis, gene ontology analysis, Kyoto encyclopedia of genes and genomes analysis, gene set enrichment analyses, and correlation analysis were performed to explore the expression and prognostic implications of glycolysis-related genes. We effectively integrated 4 genes to construct a prognostic model of shorter survival in the high-risk versus low-risk group. The prognostic model showed promising predictive value and may be an integral part of the prognosis of BC. The survival analysis and receiver operating characteristic curves suggested that the signature showed a good predictive performance in both the The Cancer Genome Atlas training set and 2 gene expression omnibus validation sets. Multivariable analysis demonstrated that the 4-gene signature had an independent prognostic value. Furthermore, all calibration curves exhibited robust validity in prognostic prediction. We established an optimized 4-gene signature to clarify the connection between glycolysis and BC, and offered an attractive platform for risk stratification and prognosis predication of BC patients.

Effects of isolated central nervous system involvement evaluated by multiparameter flow cytometry prior to allografting on outcomes of patients with acute lymphoblastic leukemia.

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a major strategy to cure patients with acute lymphoblastic leukemia (ALL). The aim of this study was to evaluate whether isolated flow cytometry (FCM)-positive central nervous system (CNS) involvement before allo-HSCT is clinically significant. Methods: The effects of isolated FCM-positive CNS involvement prior to transplantation on the outcomes of 1406 ALL patients with complete remission (CR) were retrospectively investigated. Results: Patients were classified into isolated FCM-positive CNS involvement (n=31), cytology-positive CNS involvement (n = 43), and negative CNS involvement (n = 1332) groups. Among the three groups, the 5-year cumulative incidence of relapse (CIR) values were 42.3%, 48.8%, and 23.4%, respectively (P<0.001). The 5-year leukemia-free survival (LFS) values were 44.7%, 34.9%, and 60.8%, respectively (P<0.001). Compared with the negative CNS group (n=1332), the 5-year CIR of the pre-HSCT CNS involvement group (n=74) was higher (46.3% vs. 23.4%, P<0.001], and the 5-year LFS was inferior (39.1% vs. 60.8%, P<0.001). Multivariate analysis indicated that four variables, T-cell ALL, in second complete remission or beyond (CR2+) at HSCT, pre-HSCT measurable residual disease positivity, and pre-HSCT CNS involvement, were independently associated with a higher CIR and inferior LFS. A new scoring system was developed using the following four variables: low-risk, intermediate-risk, high-risk, and extremely high-risk groups. The 5-year CIR values were 16.9%, 27.8%, 50.9%, and 66.7%, respectively (P<0.001), while the 5-year LFS values were 67.6%, 56.9%, 31.0%, and 13.3%, respectively (P<0.001). Conclusion: Our results suggest that ALL patients with isolated FCM-positive CNS involvement are at a higher risk of recurrence after transplantation. Patients with pre-HSCT CNS involvement had higher CIR and inferior survival outcomes.

CSRP2 transcript levels after consolidation therapy increase prognostic prediction ability in B-cell acute lymphoblastic leukaemia.

Quantification of measurable residual disease (MRD) correlates with the risk of leukemia recurrence in adults with B-cell acute lymphoblastic leukemia (ALL). However, it remains unknown whether collecting data on cysteine and glycine-rich protein 2 (CSRP2) transcript levels, after completing the second course of consolidation, improves prognosis prediction accuracy. A total of 204 subjects with B-cell ALL were tested for CSPR2 transcripts after completing the second course of consolidation using quantitative real-time polymerase chain reaction (qRT-PCR) and divided into high (N = 32) and low (N = 172) CSRP2 expression cohorts. In multivariable analyses, subjects with high expression of CSRP2 had a higher 5-year cumulative incidence of relapse (CIR) (hazard ratio [HR] = 2.57, 95% confidence interval [CI] 1.38-4.76; P = 0.003), lower 5-year relapse-free survival (RFS) (HR = 3.22, 95% CI 1.75-5.93; P < 0.001), and overall survival (OS) (HR = 4.59, 95% CI 2.64-7.99; P < 0.001) in the whole cohort, as well as in the multi-parameter flow cytometry (MPFC) MRD-negative cohort (for CIR, HR = 2.70, 95% CI 1.19-6.12; for RFS, HR = 4.37, 95% CI 1.94-9.85; for OS, HR = 4.90, 95% CI 2.43-9.90; all P < 0.05). Prognostic analysis showed that allogeneic hematopoietic stem cell transplantation (allo-HSCT) could significantly improve the prognosis of patients with high CSRP2 expression (allo-HSCT vs chemotherapy: 5-year CIR, 52% vs 91%; RFS, 41% vs 9%; OS, 38% vs 20%; all P < 0.05). Our data indicate that incorporating data from CSPR2 transcript levels to the MRD-testing at the end of the second course of consolidation therapy enhances prognosis prediction accuracy in adults with B-cell ALL.

Study on the cyclic adsorption performance of biomass composite membrane for Hg(II).

Salix psammophila wood flour /polyvinyl alcohol hydrogel composite membrane (SPPM) with high adsorption capacity and good cycle adsorption performance was prepared by wet spinning technology. The SPPM was characterised by the scanning electron microscope (SEM), specific surface area test (BET), energy dispersive spectrum (EDS) thermal gravimetric analysis (TGA), fourier transform infrared spectroscopy (FT-IR), and x-ray photoelectron spectroscopy (XPS). The results showed that the surface of SPPM is rough and porous, with good pore structure and thermal stability, and mercury ions (Hg(II)) have been successfully adsorbed on SPPM. At the same time, the effects of adsorption conditions (Hg(II) initial concentration, pH, adsorption time, and temperature) on the adsorption performance of SPPM were studied. Results from the adsorption experiment showed that the adsorption capacity of SPPM for Hg(II) can reach 426 mg/g. After four adsorption and desorption experiments, the adsorption capacity can reach 375 mg/g, which indicates that SPPM has good cycle adsorption performance. The adsorption kinetics was better described by the Pseudo-second-order kinetic, and their adsorption isotherms were fitted for the Langmuir model. The obtained results showed that SPPM is an available, economical adsorbent and was found suitable for removing Hg(II) from an aqueous solution.

Low IL7R Expression at Diagnosis Predicted Relapse in Adult Acute Myeloid Leukemia Patients With t(8;21).

Background: Acute myeloid leukemia (AML) with t(8;21) needs to be further stratified. In addition to leukemia cells, immune cells in tumor microenvironment participate in tumor initiation, growth and progression. Interleukins (ILs)/interleukin receptors (ILRs) interaction plays important roles in the antitumor immune response. IL7R is reported to be relevant to prognosis in solid tumor and acute lymphoblastic leukemia. However, the prognostic significance of IL7R in t(8;21) AML remains to be clarified. Methods: Bone marrows collected from 156 newly diagnosed t(8;21) AML patients were used for testing IL7R transcript level by TaqMan-based real-time quantitative PCR (RQ-PCR), and RNAseq were performed in 15 of them. Moreover, IL7R expression at diagnosis were measured by RQ-PCR and flow cytometry (FCM) simultaneously in other 13 t(8;21) AML patients. Results: t(8;21) AML patients had varied IL7R transcript levels and were categorized into low-expression (IL7R-L) and high-expression (IL7R-H) groups; IL7R-L was significantly associated with a lower relapse-free survival (RFS) rate (P=0.0027) and KITD816/D820 mutation (P=0.0010). Furthermore, IL7R-L was associated with a lower RFS rate in KITD816/D820 group (P=0.013) and IL7R-H/KITD816/D820 patients had similar RFS to KITN822/e8/WT patients (P=0.35). GO analysis enrichment showed that down-regulated genes were predominantly involved in the regulation of T cell and leukocyte activation, proliferation and differentiation in IL7R-L group. IL7R-L had significantly lower levels of Granzymes A/B, CCR7, CD28 and CD27 than IL7R-H group (all P<0.05). FCM analysis showed IL7R protein was primarily expressed in CD4+ T and CD8+ T cell subset. A significant association was found between the transcript level of IL7R and the percentage of CD8+ T cells in nucleated cells (P=0.015) but not CD4+ T cells (P=0.47). Conclusion: Low IL7R transcript level of bone marrow at diagnosis predicted relapse in t(8;21) AML, which might be caused by the difference in the amount, status and function of T cells.

Immunophenotypic characteristics of ZNF384 rearrangement compared with BCR-ABL1, KMT2A rearrangement, and other adult B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: ZNF384 rearrangement has been recently identified as a new subtype of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive studies clarifying immunophenotypic features and discriminating them from non-ZNF384 in adult BCP-ALL remain scarce to date. METHODS: Flow cytometric assessments were retrospectively performed in 43 patients with ZNF384 rearrangement, 45 with BCR-ABL1, 29 with KMT2A rearrangement and 44 with other BCP-ALL in the analysis cohort. RESULTS: CD33- and CD13-positive frequencies were significantly higher in patients with ZNF384 rearrangement than in those with non-ZNF384; however, no significant difference was observed in CD10- and CD123-positive frequencies. Analysis of antigen-positive cell proportion and median fluorescence intensity (MFI) further indicated that patients with ZNF384 rearrangement had significantly lower CD10 and higher CD33, CD13, and CD123 proportion and MFI. However, compared with KMT2A rearrangement, the CD10 expression in patients with ZNF384 rearrangement was higher, with the median percentage and MFI of 36.16 (3.63-94.79)% versus 4.53 (0.03-21.00)%, and 4.50 (0.86-32.26) versus 2.06 (0.87-4.04), respectively (p < 0.0001). Furthermore, compared with BCR-ABL1 and other BCP-ALL, ZNF384 rearrangement had significantly higher CD33 and CD13 proportion and MFI (p < 0.0001 and p < 0.05, respectively). In addition, higher CD123 proportion and MFI in ZNF384 rearrangement than those in the other three groups were reported for the first time (p < 0.01). A flow cytometry scoring system, including CD10%, CD33MFI, CD13%, and CD123MFI, was proposed and verified to predict ZNF384 rearrangement with high sensitivity and specificity, that is, 76.74% and 91.53% in the analysis and 87.50% and 91.30% in the validation cohort. CONCLUSIONS: The multiparameter immunophenotypic scoring system could suggest ZNF384 rearrangement.

Prevalence and risk factors of antibodies to HLA according to different cut-off values of mean fluorescence intensity in haploidentical allograft candidates: A prospective study of 3805 subjects.

The importance of anti-HLA antibodies in transplantation settings, such as HLA-mismatched or haploidentical hematopoietic stem cell transplantation and platelet refractoriness, is widely recognized. In previous reports, it was mentioned that several cut-off values of donor-specific anti-HLA antibodies mean fluorescence intensity (MFI) were related to graft rejection in the environment of HLA mismatched stem cell transplantation and the aim of this study was to investigate the prevalence and risk factors of anti-HLA antibodies according to those cut-off values of MFI. A total of 3805 patients with hematologic disease were prospectively enrolled and analyzed. When using MFI of anti-HLA antibodies ≥500, ≥1000, ≥1500, ≥2000, ≥5000, and ≥ 10,000 as cut-off values for positivity, the prevalence of class I or II anti-HLA antibodies ranged from 4.6% to 20.2% in all cases. When the MFI cut-off value was ≥500 for positivity, multivariate analysis indicated that platelet transfusion, underlying disease, and pregnancy were the most important risk factors for the presence of anti-HLA antibodies for the total patients. Subgroup analysis according to age, gender, and underlying disease showed that pregnancy was the most important risk factor for the presence of anti-HLA antibodies. For all patients (n = 3805), when anti-HLA antibody positivity was defined according to different MFI cut-off values, including ≥1000, ≥1500, ≥2000, ≥5000, and ≥ 10,000, an association of platelet transfusion and pregnancy with anti-HLA antibodies was also demonstrated. Our results suggest that pregnancy and platelet transfusion are the main risk factors for the prevalence of anti-HLA antibodies in haploid allograft candidates, providing evidence for guiding the evaluation of anti-HLA antibodies and helping donor selection for HLA-mismatched transplant candidates.

Low EVI1 expression at diagnosis predicted poor outcomes in pediatric Ph-negative B cell precursor acute lymphoblastic leukemia patients.

Abnormally high ecotropic viral integration site 1 (EVI1) expression has been recognized as a poor prognostic factor in acute myeloid leukemia patients. However, its prognostic impact in B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains unknown. A total of 176 pediatric Ph-negative BCP-ALL patients who received at least 1 course of chemotherapy and received chemotherapy only during follow-up were retrospectively tested for EVI1 transcript levels by real-time quantitative PCR at diagnosis, and survival analysis was performed. Clinical and EVI1 expression data of 129 pediatric BCP-ALL patients were downloaded from therapeutically applicable research to generate effective treatments (TARGET) database for validation. In our cohort, the median EVI1 transcript level was 0.33% (range, 0.0068-136.2%), and 0.10% was determined to be the optimal cutoff value for patient grouping by receiver operating characteristic curve analysis. Low EVI1 expression (<0.10%) was significantly related to lower 5-year relapse-free survival (RFS) and overall survival (OS) rates (P = 0.017 and 0.018, respectively). Multivariate analysis showed that EVI1 expression <0.10% was an independent adverse prognostic factor for RFS and OS. TARGET data showed that low EVI1 expression tended to be related to a lower 5-year OS rate (P = 0.066). In conclusion, low EVI1 expression at diagnosis could predict poor outcomes in pediatric Ph-negative BCP-ALL patients receiving chemotherapy.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2021.1939818 .

Prognostic significance of TIM-3 expression pattern at diagnosis in patients with t(8;21) acute myeloid leukemia.

Acute myeloid leukemia (AML) with t(8;21) is a heterogeneous disease and needs to be stratified. Both, cancer cells and immune cells participate in tumor initiation, growth and progression and might affect clinical outcomes. TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3), an immune checkpoint molecule, is expressed not only on immune cells but also on leukemic stem cells (LSCs) in AML. This prompted us to investigate the prognostic significance of TIM-3 in t(8;21) AML. A total of 47 t(8;21) AML patients were tested for TIM-3 expression by multi-parameter flow cytometry at diagnosis. 35 of these, who received chemotherapy alone or along with allogeneic hematopoietic stem cell transplantation were followed up. The expression pattern of TIM-3 on T-cells and NK (natural killer) cells as a whole (T + NK) and LSCs were evaluated independently. High percentage of T + NK - TIM-3+ and CD34+CD38-TIM-3+ cells were significantly associated with a high 2-year cumulative incidence of relapse (CIR) (p = 0.028, 0.016). Further, concurrent high frequencies of T + NK-TIM-3+ and CD34+CD38-TIM-3+ cells at diagnosis were significantly associated with a high 2-year CIR (p < 0.0001) and this together with c-KIT D816 mutation were the independent adverse prognostic factors for relapse (hazard ratio (HR)=2.5, [95% confidence interval (CI), 1.1-6.0], p = 0.04; HR = 46.5, [95% CI, 2.7-811.5], p = 0.009). In conclusion, the expression pattern of TIM-3 on both T and NK cells and LSCs at diagnosis had prognostic significance in t (8;21) AML.