Detection and quantification of JAK2V617F copy number by droplet digital PCR versus real-time PCR.

Myeloproliferative neoplasm (MPN) usually has an adverse prognosis, progressing to acute leukemia or splanchnic vein thromboses (SVTs). Therefore, early diagnosis and intervention are significantly important. Clinically, the burden of JAK2V617F is a vital diagnostic basis, which can be detected during the early stage of MPN. Thus, an accurate and rapid detective technique is urgently required. In recent years, real-time quantitative PCR (qPCR) has primarily been applied to detect the copies of JAK2V617F, whereas droplet digital PCR (ddPCR), a novel and promising detective tool, can conduct precise and repeatable quantification of nucleic acid copies without relying on the standard curve. In our study, both qPCR and ddPCR are used to evaluate the mutation burden of JAK2V617F in a series of gradient diluted standards and clinical JAK2V617F-positive MPN patients' bone marrow samples collected, while using next-generation sequencing technology (NGS) as a contrast. With the help of statistical methods, our study concluded that ddPCR had a better performance in accuracy, sensitivity, and stability, especially in a low burden. Regarding the accuracy, ddPCR showed a better linearity (Pearson R2 = 0.9926; P < 0.0001) than qPCR (Pearson R2 = 0.9772; P < 0.0001). What is more, ddPCR showed lower intra-assay and inter-assay CVs and the limit of detection (LOD) for the series of diluted standards than qPCR, demonstrating better stability and lower LOD. In a nutshell, ddPCR is a more promising technique for the detection and quantification of JAK2V617F.

The Combination of JAK2V617F Allele Burden and WT1 Expression can Be Helpful in Distinguishing the Subtype of MPN Patients.

INTRODUCTION: Classical Philadelphia-negative myeloproliferative neoplasm (MPN) includes Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF). The JAK2V617F mutation is part of the major criteria for diagnosis of MPN. WT1 is reported to be highly overexpressed in most hematological malignancy. Our aim was to explore the combination value of JAK2V617F allele burden and WT1 expression in distinguishing the subtype of MPN patients. METHODS: Allele specific real-time quantitative fluorescence PCR (AS-qPCR) was conducted to detect JAK2V617F allele burden. WT1 expression was assessed by RQ-PCR. Our study is a retrospective study. RESULTS: JAK2V617F allele burden and WT1 expression were different in MPN subgroups. The expression of WT1 in PMF and PV is higher than in ET. JAK2V617F allele burden in PMF and PV is also higher than in ET. ROC analysis indicated that combination of JAK2V617F allele burden and WT1 expression to discriminate ET and PV, ET and PMF, PV and PMF is 0.956, 0.871, 0.737 respectively. Furthermore, their ability to distinguish ET patients with high Hb levels from PV patients with high platelet counts is 0.891. CONCLUSIONS: Our data revealed that combination of JAK2V617F allele burden and WT1 expression is useful in distinguishing the subtype of MPN patients.

miR-638 regulates differentiation and proliferation in leukemic cells by targeting cyclin-dependent kinase 2.

MicroRNAs have been extensively studied as regulators of hematopoiesis and leukemogenesis. We identified miR-638 as a novel regulator in myeloid differentiation and proliferation of leukemic cells. We found that miR-638 was developmentally up-regulated in cells of myeloid but not lymphoid lineage. Furthermore, significant miR-638 down-regulation was observed in primary acute myeloid leukemia (AML) blasts, whereas miR-638 expression was dramatically up-regulated in primary AML blasts and leukemic cell lines undergoing forced myeloid differentiation. These observations suggest that miR-638 might play a role in myeloid differentiation, and its dysregulation may contribute to leukemogenesis. Indeed, ectopic expression of miR-638 promoted phorbol 12-myristate 13-acetate- or all-trans-retinoic acid-induced differentiation of leukemic cell lines and primary AML blasts, whereas miR-638 inhibition caused an opposite phenotype. Consistently, miR-638 overexpression induced G1 cell cycle arrest and reduced colony formation in soft agar. Cyclin-dependent kinase 2 (CDK2) was found to be a target gene of miR-638. CDK2 inhibition phenotypically mimicked the overexpression of miR-638. Moreover, forced expression of CDK2 restored the proliferation and the colony-forming ability inhibited by miR-638. Our data suggest that miR-638 regulates proliferation and myeloid differentiation by targeting CDK2 and may serve as a novel target for leukemia therapy or marker for AML diagnosis and prognosis.

The role of leukocytes in myeloproliferative neoplasm thromboinflammation.

Classic myeloproliferative neoplasms lacking the Philadelphia chromosome are stem cell disorders characterized by the proliferation of myeloid cells in the bone marrow and increased counts of peripheral blood cells. The occurrence of thrombotic events is a common complication in myeloproliferative neoplasms. The heightened levels of cytokines play a substantial role in the morbidity and mortality of these patients, establishing a persistent proinflammatory condition that culminates in thrombosis. The etiology of thrombosis remains intricate and multifaceted, involving blood cells and endothelial dysfunction, the inflammatory state, and the coagulation cascade, leading to hypercoagulability. Leukocytes play a pivotal role in the thromboinflammatory process of myeloproliferative neoplasms by releasing various proinflammatory and prothrombotic factors as well as interacting with other cells, which contributes to the amplification of the clotting cascade and subsequent thrombosis. The correlation between increased leukocyte counts and thrombotic risk has been established. However, there is a need for an accurate biomarker to assess leukocyte activation. Lastly, tailored treatments to address the thrombotic risk in myeloproliferative neoplasms are needed. Therefore, this review aims to summarize the potential mechanisms of leukocyte involvement in myeloproliferative neoplasm thromboinflammation, propose potential biomarkers for leukocyte activation, and discuss promising treatment options for controlling myeloproliferative neoplasm thromboinflammation.

Enhancer of Zeste Homolog 2 Inhibitor SHR2554 in Relapsed or Refractory Peripheral T-cell Lymphoma: Data from the First-in-Human Phase I Study.

PURPOSE: Patients with peripheral T-cell lymphomas (PTCL) in the relapsed or refractory (r/r) setting have only a limited number of therapies available, and the prognosis is extremely poor. SHR2554 is an oral inhibitor against EZH2, a rational therapeutic target for lymphomas. PATIENTS AND METHODS: This was a multicenter, two-part, phase I study of SHR2554 in r/r mature lymphoid neoplasms. In part I, 350 mg twice daily was established as the recommended phase II dose (RP2D) based on the findings during dose escalation and expansion; subsequently, selected lymphoma subtypes were recruited in clinical expansion cohorts to receive SHR2554 at RP2D. Here, we provide an in-depth assessment of SHR2554 at RP2D in subpopulation with r/r PTCL. RESULTS: Twenty-eight patients were included for analysis (17 angioimmunoblastic T-cell lymphoma and 11 not otherwise specified). Eighteen (64%) patients had received ≥2 lines of previous anticancer therapies. The objective response rate was 61% [95% confidence interval (CI), 41-78]. Responses were still ongoing in 59% (10/17) of the responders; estimated median duration of response was 12.3 months (95% CI, 7.4-not reached). Median progression-free survival was 11.1 months (95% CI, 5.3-22.0), and 12-month overall survival rate was 92% (95% CI, 72-98). The most common grade 3 or 4 treatment-related adverse events were decreased platelet count [nine (32%)] as well as decreased white blood cell count, decreased neutrophil count, and anemia [four (14%) for each]. No treatment-related deaths were reported. CONCLUSIONS: This extended follow-up analysis further supports SHR2554 as a therapeutic opportunity for patients with r/r PTCL.

A new dosing regimen of ropeginterferon alfa-2b is highly effective and tolerable: findings from a phase 2 study in Chinese patients with polycythemia vera.

Ropeginterferon alfa-2b represents a new-generation pegylated interferon-based therapy and is administered every 2-4 weeks. It is approved for polycythemia vera (PV) treatment in the United States and Europe with a starting dose of 100 µg (50 µg for patients receiving hydoxyurea) and intra-patient dose titrations up to 500 µg at 50 µg increments, which took approximately 20 or more weeks to reach a plateau dose level. This study aimed to assess ropeginterferon alfa-2b at an alternative dosing regimen with a higher starting dose and quicker intra-patient dose titrations, i.e., the 250-350-500 µg schema, in 49 Chinese patients with PV with resistance or intolerance to hydroxyurea. The primary endpoint of the complete hematologic response rate at treatment weak 24 was 61.2%, which was notably higher than 43.1% at 12 months with the approved dosing schema. The JAK2V617F allele burden decreased from baseline to week 24 (17.8% ± 18.0%), with one patient achieving a complete molecular response. Ropeginterferon alfa-2b was well-tolerated and most adverse events (AEs) were mild or moderate. Common AEs included alanine aminotransferase and aspartate aminotransferase increases mostly at grade 1 or 2 levels. Patients did not present with jaundice or significant bilirubin level increase. No grade 4 or 5 AEs occurred. Seven patients (14.3%) experienced reversible, drug-related grade 3 AEs. No AEs led to treatment discontinuation. Ropeginterferon alfa-2b at the 250-350-500 µg regimen is highly effective and well-tolerated and can help patients achieve greater and rapid complete hematologic and molecular responses.Clinical Trial Registration: This trial is registered at ClinicalTrials.gov (Identifier: NCT05485948) and in China (China National Medical Products Administration Registration Number: CTR20211664).

Efficacy and safety of QL0911 in adult patients with chronic primary immune thrombocytopenia: A multicenter, randomized, double-blind, placebo-controlled, phase III trial.

Objective: QL0911, a recombinant human thrombopoietin mimetic peptide-Fc fusion protein, is a romiplostim (Nplate®) biosimilar used to treat primary immune thrombocytopenia (ITP). This phase III study aimed to assess the efficacy and safety of QL0911 in adult patients with chronic primary ITP over a 24-week treatment period. Methods: We conducted a double-blind, placebo-controlled, phase III study in patients diagnosed with primary ITP for at least 12 months who had received at least one first-line ITP treatment with no response or recurrence after treatment, or who relapsed after splenectomy at 44 sites in China. Patients were randomly allocated (2:1 ratio) to QL0911 or placebo injection subcutaneously once weekly at an initial dose of 1 µg/kg for 24 weeks. The doses were adjusted to maintain the target platelet counts from 50 × 109/L to 200 × 109/L. Patients and investigators were blinded to the assignment. The primary endpoints were the proportion of patients who achieved a durable platelet response at week 24 (platelet count, ≥ 50 × 109/L during 6 of the last 8 weeks of treatment) and safety. The study was registered at ClinicalTrials.gov (NCT05621330). Results: Between October 2019 and December 2021, 216 patients were randomly assigned (QL0911,144; placebo,72). A durable platelet response was achieved by significantly more patients in the QL0911 group (61.8%, 95% CI: 53.3-69.8; P < 0.0001) than in the placebo group (0%). The mean duration of platelet responses was 15.9 (SE: 0.43) weeks with QL0911, and 1.9 (SE:0.26) week with placebo. Consistent results were achieved in subgroup analyses categorized by baseline splenectomy status (yes/no), concomitant ITP treatment (yes/no), and baseline platelet count (≤ 10 × 109/L, > 10 × 109/L, ≤ 20 × 109/L, > 20 × 109/L, and < 30 × 109/L). The incidence of TEAEs was comparable between the QL0911 and the placebo groups (91.7% and 88.9%, respectively). The most common adverse events overall were ecchymosis (28.5% for QL0911 vs. 37.5% for placebo), upper respiratory tract infections respiratory tract infections (31.9% for QL0911 vs. 27.8% for placebo), and gingival bleeding (17.4% for QL0911 vs. 26.4% for placebo). Conclusion: QL0911 was well-tolerated and increased and maintained platelet counts in adults with ITP. QL0911, a biosimilar to romiplostim (Nplate®), may be a novel treatment option for patients with ITP who have failed or relapsed from first-line treatment in China. Ongoing studies will provide further data on long-term efficacy and safety in such patient populations.

[Effect of calcium ionophore A23187 plus IFN-γ on dendritic cells derived from peripheral blood mononuclear cells].

OBJECTIVE: To explore the effect of calcium ionophore (CI) A23187 plus IFN-γ on dendritic cells (DC) from healthy human peripheral blood mononuclear cells (PBMNC). METHODS: PBMNC from healthy donors were treated with GM-CSF plus IL-4, A23187, and A23187 plus IFN-γ, respectively. After culture for 72 h, the change of cellular morphology was observed under light microscope and electron microscope. Surface markers on DC were analyzed by flow cytometry. MTT colorimetry was used to detect the proliferation of allogeneic T cells. Plasma concentrations of IL-12 and IFN-γ were measured by ELISA. RESULTS: PBMNC treated with A23187 plus IFN-γ for 72 h presented DC with typical morphology effectively. The surface markers CD40, CD83, and CD86 were obviously increased in group A23187 plus IFN-γ (P<0.01), but decreased in CD1a (P<0.01). In addition, it evidently stimulated the proliferation of allogeneic T cells. The levels of IL-12 and IFN-γ were significantly increased compared with other groups (P<0.01). CONCLUSION: A23187 plus IFN-γ can effectively enhance marked transformation of PBMNC into DC.

SHR2554, an EZH2 inhibitor, in relapsed or refractory mature lymphoid neoplasms: a first-in-human, dose-escalation, dose-expansion, and clinical expansion phase 1 trial.

BACKGROUND: Dysregulation of EZH2 has a crucial role in lymphomagenesis. We did a first-in-human study to assess the safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity of SHR2554, an oral EZH2 inhibitor, in patients with relapsed or refractory mature lymphoid neoplasms, including B-cell lymphomas, T-cell lymphomas, and classical Hodgkin lymphoma. METHODS: This was a multicentre, dose-escalation, dose-expansion, and clinical expansion phase 1 study done at 13 hospitals in China. Eligible patients had histologically or cytologically confirmed mature lymphoid neoplasms that had relapsed or were refractory to standard systemic therapies or had no standard-of-care. The study included a dose-escalation phase, at doses of SHR2554 from 50 mg to 800 mg twice daily; a dose-expansion phase, at two selected doses; and a subsequent clinical expansion phase at the recommended phase 2 dose in selected tumours. Primary endpoints were the safety, maximum tolerated dose, and recommended phase 2 dose. Objective response rate was a secondary endpoint. Safety and activity were assessed in all patients who received at least one dose of SHR2554 and had at least one post-baseline evaluation. This study is registered with ClinicalTrials.gov, NCT03603951, and follow-up is ongoing. FINDINGS: Between Aug 14, 2018, and July 13, 2021, 113 patients received SHR2554. At data cutoff (Sept 10, 2021), the median follow-up duration was 7·0 months (IQR 3·7-12·0). 71 (63%) patients were men and 42 (37%) were women, 110 (97%) were of Han ethnicity and 3 (3%) of other ethnicities, and 53 (47%) had received three or more lines of previous anticancer therapies. Dose-limiting toxicities occurred in two (67%) of three patients who received 400 mg SHR2554 twice daily and one (17%) of six patients who received 350 mg SHR2554 twice daily. The maximum tolerated dose and recommended phase 2 dose was determined to be 350 mg twice daily. The most common grade 3 or 4 treatment-related adverse events in all 113 patients were decreased platelet count (20 [18%]), decreased neutrophil count (ten [9%]), decreased white blood cell count (nine [8%]), and anaemia (seven [6%]). 18 (16%) patients had serious treatment-related adverse events. Two patients (2%) died due to treatment-related adverse events: one (1%) due to skin infection and toxic epidermal necrolysis and one (1%) due to respiratory failure. 107 (95%) of the 113 enrolled patients had post-baseline assessments for tumour response and were included in the activity analysis. 46 (43%; 95% CI 33-53) of these 107 patients had an overall response. INTERPRETATION: SHR2554 showed an acceptable safety profile and promising antitumour activity in patients with relapsed or refractory lymphomas, providing evidence for future investigations. FUNDING: Jiangsu Hengrui Pharmaceuticals. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Reduced miR-16 levels are associated with VEGF upregulation in high-risk myelodysplastic syndromes.

Objective: Overexpression of vascular endothelial growth factor (VEGF), a major angiogenic factor, was found in myelodysplastic syndromes (MDS) and showed different expression statuses in different risk groups of MDS. We aimed to investigate the possible role of microRNA (miR)-15a and miR-16 on the regulation of VEGF expression and their effect on angiogenesis in lower- and higher-risk MDS. Methods: We studied peripheral blood and bone marrow samples of MDS patients or several leukaemia and MDS cell lines by enzyme-linked immunosorbent assay, immunohistochemical staining, immunofluorescence and quantitative PCR for expression levels of VEGF, miR-15a and miR-16. MiRNA transfection and Luciferase reporter assays were conducted to investigate whether VEGF is a target of miR-16. Migration and tube formation assays were performed in cells exposed to medium from cells with overexpressed or knockdown miR-16. Results: It showed a significantly lower level of miR-16 in higher-risk MDS patients, while the VEGF levels were upregulated. Inverse correlation between VEGF and miR-16 were determined in cells lines including SKM-1, THP-1, and K562 cells. Overexpression of miR-16 in SKM-1 cells resulted in reduced VEGF secretion and cell protein levels. Direct binding of miR-16 to the 3' untranslated region (3'-UTR) of VEGF was confirmed by luciferase reporter assays. The migration and tube formation of human umbilical vein endothelial cells decreased in the presence of medium from SKM-1 cells with overexpressed miR-16. Conclusion: These data suggest that miR-16 may play a role in angiogenesis in higher-risk MDS by targeting VEGF and therefore modulating MDS progression. MiR-16 might be a novel therapeutic target in higher-risk MDS.

Efficacy and Safety of the Biosimilar IBI301 Plus Standard CHOP (I-CHOP) in Comparison With Rituximab Plus CHOP (R-CHOP) in Patients with Previously Untreated Diffuse Large B-Cell Lymphoma (DLBCL): A Randomized, Double-Blind, Parallel-Group, Phase 3 Trial.

INTRODUCTION: Patients with diffuse large B-cell lymphoma (DLBCL) have limited access to rituximab. IBI301 is a recombinant chimeric murine/human anti-CD20 monoclonal antibody and is a candidate biosimilar to rituximab. This study aimed to assess the therapeutic equivalence of IBI301 and rituximab in previously untreated patients with diffuse large B-cell lymphoma (DLBCL). METHODS: This multicenter, randomized, double-blind, parallel-group, phase 3 trial compared IBI301 and rituximab, both plus the chemotherapy of doxorubicin, cyclophosphamide, vindesine, and prednisone (CHOP), was conducted in 68 centers across China. Eligible patients with untreated CD20 positive (CD20+) DLBCL randomly received IBI301 (375 mg/m2) plus the standard CHOP or rituximab (375 mg/m2) plus the standard CHOP for six cycles of a 21-day cycle. The primary end point was the overall remission rate (ORR). Efficacy equivalence was defined if 95% CIs for the ORR difference between the two groups were within a ± 12.0% margin. RESULTS: Between August 22, 2016, and September 5, 2018, 419 patients were randomly allocated into the IBI301 group (N = 209) and rituximab group (N = 210). In the full analysis set, the ORR was 89.9% and 93.8% in the IBI301 and rituximab groups, respectively, and the ORR difference was -3.9% (95% CI - 9.1%-1.3%), falling within a ± 12.0% margin. The occurrences of treatment-emergent adverse events (TEAEs) (100% vs. 99.0%) and AEs of grade ≥ 3 (87.1% vs. 83.3%) were similar in the two groups (P > 0.05). CONCLUSIONS: IBI301 had a non-inferiority efficacy and a comparable safety compared with rituximab. IBI301 plus CHOP could be suggested as a candidate treatment regimen for untreated patients with CD20+ DLBCL. TRIAL REGISTRATION: This trial is registered on ClinicalTrials.gov (NCT02867566).

Reversal of multidrug resistance by curcumin through FA/BRCA pathway in multiple myeloma cell line MOLP-2/R.

Most patients with multiple myeloma (MM) will relapse eventually due to the acquired multidrug resistance (MDR). The objective of this study was to explore the reversal effect of curcumin on the MDR of human MM cell line, MOLP-2/R, and analyze the role of Fanconi anemia (FA)/BRCA pathway in this process. MOLP-2/R was selected by stepwise exposure of parental MOLP-2 cells to increasing concentrations of melphalan. The MTT assay was used to detect the reversal ratio of curcumin. The FANCD2 monoubiquitination expression was detected by western blotting to explore the role of FA/BRCA pathway. Cell cycle, apoptosis, and intracellular drug concentration were analyzed by flow cytometry. The results indicated that combination of melphalan with curcumin had stronger effects on the proliferation inhibition, inducement of apoptosis, G2/M phase arrest, and enhancement of intracellular drug concentration than melphalan alone in MOLP-2/R cells. These effects were accompanied with inhibition of FA/BRCA pathway by down regulation of FANCD2 protein monoubiquitination in a dose-dependent manner. In conclusion, curcumin reversed multidrug resistance of MOLP-2/R through inhibition of FA/BRCA pathway. The possible mechanisms include (1) reduction of DNA damage repair and stimulation of apoptosis of tumor cells through inhibition of FA/BRCA pathway, which is important for DNA repair, and (2) achievement of high concentration in target cells. Curcumin may be a safe reversal agent of multidrug resistance with low-dose DNA cross-linking agents.

The effect of miR-223 on cellular behaviour in non-5q myelodysplastic syndromes through targeting RPS14.

Myelodysplastic syndromes (MDS) are characterised by impaired haematopoiesis and a high risk of leukaemic transformation. A decrease in RPS14 expression in non-5q MDS patients was confirmed by immunohistochemical analyses of MDS bone marrow biopsies. To determine the cause of RPS14 reduction in non-5q MDS, we analysed the 3'-UTR of RPS14 and demonstrated that miR-223 binds to the 3'-UTR of RPS14 by bioinformatics-based approach combined with the luciferase reporter assay. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we observed a significantly increased expression of miR-223 in CD34+ cells and SKM-1 cells derived from non-5q MDS patients in vitro and demonstrated a correlation between miR-223 levels and red blood cell counts. Exogenous miR-223 expression in SKM-1 cells could also inhibit RPS14 expression. In functional studies, overexpression of miR-223 was shown to promote cell proliferation and inhibit cell apoptosis in SKM-1 cells, and to impair erythroid differentiation in haemin-induced K562 cells. Taken together, our results revealed that the overexpression of miR-223 in MDS is closely associated with cell transformation and erythroid differentiation arrest, which is most likely mediated by targeting RPS14.

SF3B1 Mutation but Not Ring Sideroblasts Identifies a Specific Group of Myelodysplastic Syndrome-Refractory Cytopenia With Multilineage Dysplasia.

BACKGROUND: Integrating the proportion of ring sideroblasts and SF3B1 mutation status is required for diagnosis of sideroblastic subgroups in myelodysplastic syndrome (MDS) as proposed by the World Health Organization 2016 classification. However, the clinical implications of SF3B1 mutation and ring sideroblasts in MDS-refractory cytopenia with multilineage dysplasia (MDS-RCMD) remain unclear. PATIENTS AND METHODS: Clinical and laboratory features in 238 MDS-RCMD patients were retrospectively analyzed, and the prognostic significance of SF3B1 mutation and ring sideroblasts on overall survival and leukemia-free survival in total MDS-RCMD patients and different subgroups stratified by the percentage of ring sideroblasts or SF3B1 mutation status were evaluated. RESULTS: MDS-RCMD patients with ring sideroblasts ≥ 15% showed a significantly higher prevalence of SF3B1 mutation compared to ring sideroblasts 5%-14% or ring sideroblasts < 5% (75.6% vs. 15.1% vs. 6.4%, P < .001). In multivariate analysis, SF3B1 mutation was associated with a significantly prolonged survival (hazard ratio [HR] = 0.430, P = .013) and reduced leukemic transformation (HR = 0.174, P = .021) in total MDS-RCMD patients, while ring sideroblasts showed no independent effect on either survival or leukemic transformation. There were no significant differences in clinical characteristics or survival between MDS-RCMD patients with ring sideroblasts ≥ 15% and ring sideroblasts 5%-14% in the presence of SF3B1 mutation. Furthermore, SF3B1 mutation showed an independent prognostic effect on overall survival in MDS-RCMD patients with ring sideroblasts 5%-14% (HR = 0.195, P = .046). CONCLUSION: SF3B1 mutation, not the presence of ring sideroblasts, identifies a distinct subtype and showed independent prognostic value on survival and leukemia transformation in MDS-RCMD patients.

Current Status and Perspective Biomarkers in AFP Negative HCC: Towards Screening for and Diagnosing Hepatocellular Carcinoma at an Earlier Stage.

Hepatocellular carcinoma (HCC) is one of the most malignant cancer with high morbidity and mortality which lead to a serious burden to society. AFP (alpha-fetoprotein) is the most widely used serum biomarker to detect HCC worldwide. However, no AFP elevation have been found in many HCC and AFP analysis can't be used to screen HCC in these cases. Currently, many studies have been carried out to find reliable biomarker in diagnosing AFP-negative HCC. Such biomarker would help the diagnosis of AFP-negative HCC, ensuring the timely initiation of treatment. In this review, we highlight the important role of biomarkers that can differentiate AFP-negative HCCs, and discuss their potential clinical applications as biomarkers for the diagnosis of AFP-negative HCC.

Cytokines frequently implicated in myeloproliferative neoplasms.

Classical myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). MPN has been defined as a chronic inflammation-driven tumor model. It is clear that there is a close link between chronic inflammation and MPN pathogenesis. Several studies have demonstrated cytokine profiles in MPN patients. Other studies have used cell lines or animal models aiming to clarify the underlying mechanism of cytokines in the pathogenesis of MPN. However, important questions remain: (1) among all these cytokines, which are more predictive? and (2) which are more critical? In this review, we summarize cytokines that have been investigated in MPN and highlight several cytokines that may be more significant in MPN. We suggest that cytokines are more critical in PMF than PV or ET. These cytokines include IL-1ß, TNF-α, IL-6, IL-8, VEGF, PDGF, IFNs and TGF-ß, all of which should be more closely investigated in MPN. Based on our extensive literature search, several key factors have emerged in our understanding of MPN: first, TNF-α could correlate with MPN progression including PMF, PV and ET. IL-1ß plays a role in PMF progression, while it showed no relation with PV or ET. Second, IL-8 could be a prognostic factor for PMF, and IL-6 could be important for MPN progression. Third, VEGF and PDGF play an indirect role in MPN development and their inhibitors could be effective. Fourth, different subtypes of IFNs could have different effects in MPN. Finally, TGF-ß is closely linked to MF, although the data are inconsistent. Agents that have targeted these cytokines described above are already in clinical trials, and some of them have even been used to treat MPN patients. Taken together, it will be critical to continue to investigate the precise role of these cytokines in the pathogenesis and progression of MPN.

A hospital based retrospective study of factors influencing therapeutic leukapheresis in patients presenting with hyperleukocytic leukaemia.

Therapeutic leukapheresis is a rapid and effective method to reduce early mortality of patients with hyperleukocytic leukaemia (HLL). However, few studies on factors influencing the efficiency have been reported. In this study, 67 cases who underwent leukapheresis were retrospectively analysed and factors related to the collection efficiency of leukapheresis (CEWBC) were also evaluated. Paired t test showed that there was a significant decrease in statistics of white blood cell (WBC) counts after apheresis. The results of two independent samples nonparametric test suggested that WBC counts, platelet (PLT) counts, haematocrit (HCT), hemoglobin (HGB), serum chlorine (Cl) and globulin (GLB) before leukapheresis correlated with the CEWBC. Multiple linear regression analysis with background stepwise variable selection indicated that only WBC and HCT before leukapheresis had an influence on CEWBC significantly. Kaplan-Meier analysis and Cox regression model indicated that lymphocyte (LY) and mean corpuscular hemoglobin (MCH) pre-apheresis as independent factors significantly affected the prognostic survival of patients with HLL. Moreover, platelets and red blood cell were contaminated in the product of leukapheresis. It is an urgent problem to be solved in order to realise higher efficacy and higher purity of WBC collection to improve the survival of patients with HLL through optimising instruments.

ASXL1 mutations in Chinese patients with essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by thrombotic and hemorrhagic events. The association of clinical characteristics of Chinese ET patients and additional sex combs like 1 (ASXL1) mutations in these patients has remained to be elucidated. In the present study, 72 newly diagnosed Chinese ET patients were enrolled to determine ASXL1 mutations. Mutations in ASXL1, Janus kinase (JAK)2, calreticulin (CALR) and myeloproliferative leukemia (MPL) genes were detected using Sanger sequencing, and data were statistically analyzed. The frequencies of ASXL1, JAK2 V617F, CALR and MPL W515 mutations in ET patients were 19.4% (14/72), 29.2% (21/72), 31.9% (23/72) and 0% (0/72), respectively. Of note, 28 ET patients (38.9%) were negative for JAK2, CALR and MPL mutations; these patients were classified as triple-negative (TN). The frequency of ASXL1 mutations in patients with JAK2 V617F, CALR and TN mutations was 23.8% (5/21), 21.7% (5/23) and 14.3% (4/28), respectively. ASXL1-mutant patients exhibited significant propensities for thrombotic events compared with the ASXL1 wild-type (wt) cohort (42.9 vs. 12.1%; P=0.021). In addition, JAK2 V617F-mutant patients had a higher mean age compared with CALR-mutant (64.76 vs. 52.96 years; P=0.008) or TN patients (64.76 vs. 51.14 years; P=0.002). Furthermore, more white blood cells in the peripheral blood (PB) were observed in JAK2 V617F-mutant patients compared with those in TN patients (12.40 vs. 8.20×109/l; P=0.02). In addition, CALR-mutant patients exhibited more platelets (PLT) in PB than JAK2 V617F-mutant patients (787.91 vs. 562.17×109/l; P=0.047). TN patients had a significantly lower incidence of clinical symptoms, including dizziness, palpitation and chest congestion compared with CALR- or JAK2 V617F-mutant patients (14.1 vs. 39.1%; P=0.043 and 14.1 vs. 38.1%; P=0.050). No significant difference in progression-free survival was observed between ASXL1-mutant and ASXL1-wt patients (P=0.590). In conclusion, ASXL1-mutant ET patients are prone to experiencing thrombotic events. There was no significant difference in the occurrence of thrombotic events among CARL-mutant, JAK2 V617F-mutant and TN patients. Furthermore, ASXL1-mutant/TN patients exhibited a higher number of PLT than ASXL1/JAK2 V617F-double mutant patients. Therefore, ASXL1 mutations may be a risk factor for the occurrence of thrombotic events in ET patients.

Prognostic evaluation of ALIP and CD34 immunostaining in IPSS-R subgroups of myelodysplastic syndromes.

In order to evaluate the prognostic value of abnormal localisation of immature precursors (ALIP) and CD34 immunostaining in myelodysplastic syndromes (MDS), bone marrow histopathological features in 187 MDS patients were retrospectively analysed and the prognostic significance of ALIP and CD34 immunostaining on overall survival (OS) and progression to leukaemia-free survival (PFS) in total patients and different Revised-International Prognostic Scoring System (IPSS-R) subgroups were evaluated. In univariate analysis, age ≥60, ALIP, ≥5% CD34+ cells, CD34+ clusters and IPSS-R subgroups were associated with shorter OS (p = 0.027, p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, respectively) and PFS (p = 0.029, p = 0.006, p = 0.001, p < 0.0001, p < 0.0001, respectively). Haemoglobin level had a significant impact on OS (p < 0.0001) but not on PFS (p = 0.054). In multivariate analysis, ALIP, haemoglobin level, ≥5% CD34+ cells, CD34+ clusters and IPSS-R subgroups had independent influence on OS (p = 0.012, p < 0.0001, p = 0.010, p < 0.0001, p < 0.0001, respectively), while only CD34+ clusters and IPSS-R subgroups had independent influence on PFS (p < 0.0001, p = 0.016, respectively). In different IPSS-R subgroups, ALIP could maintain its prognostic impact in lower IPSS-R risk subgroups, while ≥5% CD34+ cells and CD34+ clusters had significant prognostic value in both lower and intermediate-higher IPSS-R risk subgroups. Therefore, CD34+ clusters showed more important prognostic impact on survival and progression to leukaemia.