Changxin Yin

A report on the co-occurrence of JAK2V617F and CALR mutations in myeloproliferative neoplasm patients

Dear Editor, Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells, with Janus kinase 2 (JAK2) and calreticulin (CALR) mutations being recurrent and mutually exclusive in patients with MPNs. CALR mutations have been reported in a significant percentage of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) that lack a JAK2V617F mutation. However, there have been three recent reports of one PMF and two ET patients who have concurrent JAK2V617F and CALR exon 9 mutations [1–3]. There is no known research report on polycythemia vera (PV) patients with the double gene mutations thus far. Here, we report for the first time a single PV patient who carries concomitant CALR and JAK2V617F mutations and one ET patient carrying the double gene mutations. Interestingly, these two patients had a good response to interferon alfa treatment. Case 1: In August 2012, a 65-year-old male patient presented to our institution with a year-long history of increased weakness and fatigue with splenomegaly and no adenopathy. The peripheral blood count showed polycythemia (hemoglobin 205 g/L, hematocrit 56 %), thrombocythemia (platelet count 508×10/L), a white blood cell count (WBC) of 7.8×10/μL (neutrophils 57 %, lymphocytes 39 %, monocytes 4 %), and low erythropoietin levels (2.96 IU/L). A bone marrow biopsy revealed hypercellularity without fibrosis. Fluorescence in situ hybridization (FISH) revealed a lack of both BCR/ABL and FIPL/PDGFRα fusion genes, and the patient’s karyotype was normal (46, XY[20]). We detected a homozygous JAK2V617F mutation (Fig. 1b) by real-time quantitative polymerase chain reaction as well as an uncommon variant of a 3 bp (AAG) deletion in exon 9 of CALR by bidirectional sequencing. The latter mutation has been classified as c.1095-1097del, p.E371fs*49 [4] (Fig. 2b). The patient was diagnosed with PV and was administered with interferon alfa in October 2012 for 18 months, at which time he had a complete hematologic response without splenomegaly. This patient was still in complete remission without cytoreductive therapy as of July 2014. Case 2: In July 2013, a 63-year-old female patient was admitted to our hospital with a history of gradual elevation of platelet counts accompanied by intermittent dizziness for 3 years. The patient is well and without history of bleeding, thrombosis, cerebrovascular disease, o r any o the r occ lus ive symptoms , inc lud ing hepatosplenomegaly and neurological issues. The peripheral blood count showed a platelet count of 1350× 10/L, WBC of 15.1×10/L, and hemoglobin levels of 121 g/L. A bone marrow aspirate showed an increase in megakaryocytes with hyperlobulated nuclei. A heterozygous JAK2V617F mutation (Fig. 1c) and a 52 bp deletion in exon 9 of CALR (classified as c.1092_1143del) [5] (Fig. 2c) were detected without cytogenetic Na Xu and Li Ding contributed equally to this study and should be considered as co-first authors.

Herpesvirus-associated central nervous system diseases after allogeneic hematopoietic stem cell transplantation.

Herpesvirus infections of the central nervous system (CNS) are associated with encephalitis/myelitis and lymphoproliferative diseases in immunocompromised individuals. As of now, data of herpesvirus-associated CNS diseases in transplant recipients is limited. Hence, in this prospective study, we investigated the incidence of herpesvirus-associated CNS diseases and explored the diagnosis of these diseases in 281 allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Herpesvirus-DNA and cerebrospinal fluid (CSF) cells were sampled from 58 recipients with herpesvirus-associated diseases or with unexplainable CNS manifestations. Results showed that 23 patients were diagnosed as herpesvirus-associated CNS diseases, including 15 Epstein-Barr virus (EBV)-associated diseases (4 encephalitis and 11 lymphoproliferative diseases), 5 herpes simplex virus type 1 encephalitis, 2 cytomegalovirus encephalitis/myelitis and 1 varicella zoster virus encephalitis. The median time of diseases onset was 65 (range 22-542) days post-transplantation. The 3-year cumulative incidence of herpesvirus-associated encephalitis/myelitis and post-transplant lymphoproliferative disorder (PTLD) was 6.3% ±1.9% and 4.1% ±1.2%, respectively. Of the evaluable cases, CSF cells mainly consisted of CD19+CD20+ B cells (7/11) and had clonal rearrangement of immunoglobulin genes (3/11) in patients with CNS-PTLD. On the contrary, in patients with encephalitis/myelitis, CSF cells were comprised of different cell populations and none of the gene rearrangement was detected. Herpesvirus-associated CNS diseases are common in the early stages of allo-HSCT, wherein EBV is the most frequent causative virus. The immunophenotypic and clonal analysis of CSF cells might be helpful in the differential diagnosis between encephalitis and lymphoproliferative diseases.

Gli-1/PI3K/AKT/NF-kB pathway mediates resistance to radiation and is a target for reversion of responses in refractory acute myeloid leukemia cells

Total body irradiation combined with chemotherapy is currently the most effective procedure as a preparative myeloablative regimen. However, resistance to radiotherapy and chemotherapy in refractory acute myeloid leukemia is associated with short-time recurrence after allogeneic hematopoietic stem cell transplantation. To address this issue, we used three cell lines, HL60, HL60/ADR (adriamycin-resistant cells), and HL60/RX (a radiation-resistant cell line established from HL60 cells), as cellular models to investigate the mechanism of the Hedgehog (Hh) signaling pathway resulting in radioresistance, and the efficacy of LDE225 (an inhibitor of the Hh pathway) to enhance radiation sensitivity. Our results indicated that HL60/RX and HL60/ADR cells showed an increased in radioresistance and elevated activity of Hh pathway proteins compared with HL60 cells (P<0.001). In addition, LDE225 significantly reduced clonogenic survival with a sensitivity enhancement ratio (SER) of 1.283 for HL60/ADR and 1.245 for HL60/RX cells. The combination of LDE225 with irradiation significantly increased radiation-induced apoptosis and expression of γ-H2AX and BAK compared with single-treatment groups in both HL60/RX and HL60/ADR cells (P<0.001). In vivo, the combination of LDE225 with irradiation exerted a significant antitumor effect compared with the control and single agents in HL60/RX- and HL60/ADR-xenografted mouse models (P<0.001). Furthermore, our data obtained from western blot and IHC analyses showed that the activation of pAKT and NF-kB was reduced by LDE225 treatment in both HL60/ADR and HL60/RX cells. This demonstrates that the Gli-1/PI3K/AKT/NF-kB pathway plays a key role in resistance to radiation, and that inhibition of the Hh pathway sensitizes cells to radiation by overcoming radioresistance.

Immunophenotypes and Immune Markers Associated with Acute Promyelocytic Leukemia Prognosis

CD2+, CD34+, and CD56+ immunophenotypes are associated with poor prognoses of acute promyelocytic leukemia (APL). The present study aimed to explore the role of APL immunophenotypes and immune markers as prognostic predictors on clinical outcomes. A total of 132 patients with de novo APL were retrospectively analyzed. Immunophenotypes were determined by flow cytometry. Clinical features, complete remission (CR), relapse, and five-year overall survival (OS) rate were assessed and subjected to multivariate analyses. The CD13+CD33+HLA-DR-CD34− immunophenotype was commonly observed in patients with APL. Positive rates for other APL immune markers including cMPO, CD117, CD64, and CD9 were 68.7%, 26%, 78.4%, and 96.6%, respectively. When compared with patients with CD2− APL, patients with CD2+ APL had a significantly higher incidence of early death (50% versus 15.7%; P = 0.016), lower CR rate (50% versus 91.1%; P = 0.042), and lower five-year OS rate (41.7% versus 74.2%; P = 0.018). White blood cell (WBC) count before treatment was found to be the only independent risk factor of early death, CR failure, and five-year mortality rate. Flow cytometric immunophenotype analysis can facilitate prompt APL diagnosis. Multivariate analysis has demonstrated that WBC count before treatment is the only known independent risk factor that predicts prognosis for APL in this study population.

The hypomethylating agent decitabine prior to chemotherapy improves the therapy efficacy in refractory/relapsed acute myeloid leukemia patients

In this study, we investigated the effect of pre-treatment with demethylating agent decitabine on susceptibility to chemotherapeutic drugs in HL60/ADR, Kasumi-1 and primary AML cells. Cytotoxic effect was increased by decitabine through activation of p53 and inhibition of c-Myc, Survivin and Bcl-2. We demonstrated in clinic that combination of decitabine and HAA consisting of harringtonine, aclarubicin and cytarabine was effective and safe to treat patients with refractory, relapsed or high-risk AML. Decitabine prior to HAA regimen improved the first induction complete response rate, and significantly prolonged overall survival and disease-free survival in these patients compared with HAA alone. These findings support clinic protocols based on decitabine prior to chemotherapy to overcome resistance and improve therapeutic efficacy in AML patients.

Identification of Novel Molecular Markers for Prognosis Estimation of Acute Myeloid Leukemia: Over-Expression of PDCD7, FIS1 and Ang2 May Indicate Poor Prognosis in Pretreatment Patients with Acute Myeloid Leukemia

Numerous factors impact on the prognosis of acute myeloid leukemia (AML), among which molecular genetic abnormalities are developed increasingly, however, accurate prediction for newly diagnosed AML patients remains unsatisfied. For further improving the prognosis evaluation system, we investigated the transcripts levels of PDCD7, FIS1, FAM3A, CA6, APP, KLRF1, ATCAY, GGT5 and Ang2 in 97 AML patients and 30 non-malignant controls, and validated using the published microarray data from 225 cytogenetically normal AML (CN-AML) patients treated according to the German AMLCG-1999 protocol. Real-time quantitative polymerase chain reaction and western blot were carried out, and clinical data were collected and analyzed. High Ang2 and FIS1 expression discriminated the CR rate of AML patients (62.5% versus 82.9% for Ang2, P = 0.011; 61.4% versus 82.2% for FIS1, P = 0.029). In CN-AML, patients with high FIS1 expression were more likely to be resistant to two courses of induction (P = 0.035). Overall survival (OS) and relapse-free survival (RFS) were shorter in CN-AML patients with high PDCD7 expression (P<0.001; P = 0.006), and PDCD7 was revealed to be an independent risk factor for OS in CN-AML (P = 0.004). In the analysis of published data from 225 CN-AML patients, PDCD7 remained independently predicting OS in CN-AML (P = 0.039). As a conclusion, Ang2 and FIS1 seem related to decreased CR rate of AML patients, and PDCD7 is associated with shorter OS and RFS in CN-AML. Hence, PDCD7, Ang2 and FIS1 may indicate a more aggressive form and poor prognosis of AML.

Higher EZH2 expression is associated with extramedullary infiltration in acute myeloid leukemia.

Accumulating evidence indicates that enhancer of zeste homolog 2 (EZH2) promotes the metastatic ability of solid tumors, but the role of EZH2 in extramedullary infiltration (EMI) in acute myeloid leukemia (AML) has not been thoroughly explored. In the present study, we investigated the possible association between EZH2 and EMI. We found that the messenger RNA (mRNA) and protein expression levels of EZH2 in AML patients were both significantly higher than in idiopathic thrombocytopenic purpura (ITP) patients. Furthermore, a positive correlation between EZH2 mRNA expression and percentage of peripheral blood blasts wa s found in AML patients (r = 0.404, p = 0.009). The migratory capacities of Kasumi-1 and HL-60, which both show a high level of EZH2 expression, were markedly higher than those of U937 and KG-1α. In contrast, silencing of EZH2 resulted in reduction in proliferation and migration ability and an increase in apoptosis. The latter observation was accompanied by reduced expression of associated proteins p-ERK, p-cmyc, and matrix metalloproteinase 2 (MMP-2) and an increase in epithelial cadherin (E-cadherin). These data suggest that higher expression of EZH2 may be associated with extramedullary infiltration in acute myeloid leukemia and affect pathogenesis via activation of the p-ERK/p-cmyc/MMP-2 and E-cadherin signaling pathways.

Effect of sorafenib on the outcomes of patients with FLT3-ITD acute myeloid leukemia undergoing allogeneic hematopoietic stem cell transplantation

The objective of this study was to evaluate the effect of sorafenib on the outcomes of patients with acute myeloid leukemia (AML) with FMS‐like tyrosine kinase 3 (FLT3)–internal tandem duplication (ITD) undergoing allogeneic hematopoietic stem cell transplantation (allo‐HSCT).

The Relationship between Clinical Feature, Complex Immunophenotype, Chromosome Karyotype, and Outcome of Patients with Acute Myeloid Leukemia in China

Mixed phenotype acute leukemia (MPAL) is a complex entity expressing both lymphoid and myeloid immunophenotyping. In the present study, 47 MPAL, 60 lymphoid antigen-positive acute myeloid leukemia (Ly+AML), and 90 acute myeloid leukemia with common myeloid immunophenotype (Ly−AML) patients were investigated. We found that, in MPAL patients, there were high proportions of blast cells in bone marrow and incidence of hepatosplenomegaly, lymphadenopathy, and Philadelphia chromosome. The overall survival (OS) and relapse-free survival (RFS) in MPAL patients were significantly shorter than those in Ly+AML and Ly−AML. With regard to the patients with normal karyotype only, the OS and RFS of MPAL were significantly lower than those of the Ly+AML and Ly−AML; but there were no significant differences in OS and RFS among the patients with complex karyotype. The OS rates of 3 groups with complex karyotype were lower than those of patients with normal karyotype. In Cox multivariate analysis, complex karyotype was an independent pejorative factor for both OS and RFS. Therefore, MPAL is confirmed to be a poor-risk disease while Ly+AML does not impact prognosis. Complex karyotype is an unfavorable prognosis factor in AML patients with different immunophenotype. Mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.

Influence of initiation time and white blood cell count on the efficacy of cytotoxic agents in acute promyelocytic leukemia during induction treatment

The present study retrospectively analyzed 96 newly diagnosed acute promyelocytic leukemia (APL) patients with low-intermediate mortality risk to identify the optimum timing to initiate cytotoxic chemotherapy following all-trans retinoic acid (ATRA) administration. Based on white blood cell (WBC) at chemotherapy initiation, the patients were divided into three groups: low WBC (WBC count ≤4×109/l), intermediate WBC (WBC count >4×109/l and <15×109/l) and high WBC group (WBC count ≥15×109/l). According to the period from ATRA commencement to chemotherapy, 96 patients were further divided into two groups: ≤3 days group (chemotherapy within 3 days of ATRA) and >3 days group (chemotherapy >3 days after ATRA). Clinical effects were compared by univariate analysis and multivariate analyses. The incidence rate of differentiation syndrome (DS; also termed retinoic acid syndrome) was 0.0, 11.1 and 40.0% in the low, intermediate and high WBC groups, respectively (P<0.001); complete remission (CR) rate was 90.5, 100.0 and 73.3%, respectively (P<0.001); and the rate of early mortality (defined as fatality during induction treatment) was 4.8, 0.0 and 26.7%, respectively (P<0.001). No differences were identified in clinicolaboratory parameters between the ≤3 days and >3 days groups, except in time to achieve CR (P=0.004) and rate of bleeding related to chemotherapy (P=0.009), both being higher in the >3 days group. Multivariate analyses indicated WBC count at chemotherapy was the only independent risk factor for the occurrence of DS [P=0.002; odds ratio (OR) =1.058, 95% confidence interval (CI) =1.021-1.095] and early mortality (P=0.036; OR =1.036, 95% CI =1.002-1.070). For newly diagnosed APL patients with low-intermediate risk, chemotherapy initiation should be recommended until WBC count rises to between 4×109/l and 15×109/l during induction treatment.