Su-Jiang Zhang

GATA-2 L359 V mutation is exclusively associated with CML progression but not other hematological malignancies and GATA-2 P250A is a novel single nucleotide polymorphism

Chronic myeloid leukemia (CML) progression is characterized by occurrence of new cytogenetic and molecular abnormalities. In the previous study, we have shown the important role of GATA-2 L359 V mutation in CML progression. To further ascertain the truth of transcription factor GATA-2 in hematological malignancies, we expanded our study to GATA-2 full length by directly sequencing and applied MassARRAY assay into GATA-2 L359 V mutation analysis. Finally, no GATA-2 L359 V mutation was found in 270 acute myeloid leukemia, 30 myelodysplastic syndrome, 50 acute lymphoblastic leukemia, 12 chronic lymphocytic leukemia, 40 CML chronic phase and 286 BCR/ABL negative myeloproliferative disorders except CML blast crisis. A new variation of GATA-2 resulted in P250A change was identified, which was not found to have statistical difference between patients with hematological malignancies and healthy control. Hence, we concluded GATA-2 L359 V is exclusively associated with CML progression but not other hematological malignancies and P250A is a new single nucleotide polymorphism.

Clinical importance of different calreticulin gene mutation types in wild-type JAK2 essential thrombocythemia and myelofibrosis patients.

The Janus kinase 2 (JAK2) V617F mutation (JAK2 V617F), JAK2 exon 12 mutations and myeloproliferative leukemia virus oncogene W515L/K mutation (MPL W515L/K) have become three major molecular diagnosis criteria for myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) from 2005.1 However, diagnosing MPNs with non-mutated JAK2 and MPL remains a major diagnostic challenge.2–4 Some recent studies have reported calreticulin (CALR) gene mutations in patients with non-mutated JAK2 V617F MPNs.5–7 There are some distinct mutation types in MPN subtypes, but the differences in the clinical significance and prognosis among the different mutation types are obscure.8–10 Here, we report our data on CALR mutation in wild-type (wt) JAK2 MPN on patients. It should also be mentioned that this is undoubtedly the first report regarding CALR mutations in Chinese MPN patients. From January 2008 to December 2013, bone marrow or peripheral blood samples from 301 MPNs patients were collected in the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province, China, including ET (n=222), PV (n=37), PMF (n=33), post-ET MF (PET-MF; n=6), and post-PV MF (PPV-MF; n=3). We also obtained bone marrow samples from 174 patients with other myeloid neoplasms including: myelodysplastic syndrome (MDS; n=8), chronic myelogeneous leukemia (CML; n=55), acute myeloid leukemia (AML; n=104), and chronic myelomonocytic leukemia (CMML; n=7), as well as peripheral blood samples from 121 healthy controls. All participants provided their informed consent. Genomic PCR combined with direct and cloning sequencing was applied to screen CALR mutations. A total of 24.3% (73 of 301) patients with MPNs were found harboring CALR mutations. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of patients with ET and PMF, respectively (Figure 1A). Moreover, CALR mutations were found in 57.0% (69 of 121) ET patients with wt JAK2 and 30.8% (4 of 13) PMF patients with wt JAK2. No CALR mutation in patients with PV, PET-MF, PPV-MF (Figure 1A) was found. The CALR mutations have multiple deletions or insertions including: L367fs*46 (33 of 74; 44.6%), K385fs*47 (25 of 74; 33.8%), K368fs*51 (3 of 74; 4.1%), Q365fs*50 (3 of 74; 4.1%), E364fs*49 (2 of 74; 2.7%), K374fs*56 (2 of 74; 2.7%), L367fs*48 (1 of 74; 1.4%), Q365fs*48 (1 of 74; 1.4%), E364fs*55 (1 of 74; 1.4%), K375fs*48 (1 of 74; 1.4%), K375fs*55 (1 of 74; 1.4%), and K377fs*50 (1 of 74; 1.4%). Figure 1. (A) Frequency of CALR mutations in myeloid neoplasms and healthy control. The CALR mutation was detected in 31.1% (69 of 222) and 12.1% (4 of 33) of ET and PMF patients, respectively. Approximately 1% of patients (1 of 104) with AML were found to harbor ... These patients with MPNs were simultaneously examined for the presence of other gene mutations. PV patients were screened for JAK2 V617F and JAK2 exon 12 mutations, while ET and MF patients were screened for JAK2 V617F and MPL W515L/K mutation. Among the total 301 patients with MPNs, 52.2% (157 of 301) were found to harbor JAK2 V617F mutation. Among the 222 patients with ET and 37 patients with PV, 0.9% (2 of 222) were found to harbor MPL W515L/K mutations and 2.7% (1 of 37) to harbor JAK2 exon 12 mutation, respectively (Figure 1B). JAK2 V617F, JAK2 exon 12 mutation, MPL W515L/K mutations and CALR mutations were found exclusively in these MPNs patients. We also screened CALR mutations in 104 AML patients, 55 CML patients, 7 CMML patients, and 8 MDS patients (including 5 refractory cytopenia with multilineage dysplasis, 2 refractory anemia with excess blasts, and one refractory anemia) to investigate whether CALR mutations were present in other myeloid neoplasms. Although most of these patients had negative results, one AML patient (59-years old, male, M2 subtype) was found to harbor CALR mutation (L367fs*46) without JAK2 V617F and MPL W515L/K mutations (Figure 1A). This patient had no previous history of MPN or MDS, Fms-related tyrosine kinase 3 internal tandem duplication, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog mutation, nucleophosmin mutation. CCAAT/enhancer binding protein alpha mutation was all negative and cytogenetics analysis showed normal karyotype. In addition, no CALR mutation was detected in the 121 healthy controls (Figure 1A). For mutation types, a total of 12 distinct variants of CALR mutation, including 11 deletions and one insertion, were identified in our patients. L367fs*46, which resulted from a 52-bp deletion, and K385fs*47, which resulted from a 5-bp insertion, were the most frequent CALR mutations. The two mutations accounted for 44.6% (33 of 74) and 33.8% (25 of 74) in all cases with mutant CALR, respectively. For ET patients, the two mutations were 14.0% (31 of 222) and 10.4% (23 of 222), respectively. For PMF patients, the two mutations were 3.0% (1 of 33) and 6.1% (2 of 33), respectively. There was no significant difference in the two mutation types between patients with ET and PMF (P=0.137 and P=0.645, respectively). Moreover, the two mutations were 44.9% (31 of 69) and 33.3% (23 of 69) in CALR mutation positive ET patients (Figure 1C), as well as 25% (1 of 4) and 50% (2 of 4) in CALR mutation positive PMF patients (P=0.793 and P=0.888, respectively). There were few other mutation types in the CALR-mutated samples (Figure 1C). ET patients with mutant CALR were significantly younger (P<0.001) and had lower white blood cell (WBC) counts (P<0.001), lower hemoglobin (Hb) levels (P=0.002), and higher platelet (PLT) counts (P<0.001) than patients with JAK2 V617F. No significant difference can be identified between ET patients with mutant CALR and JAK2 V617F in terms of sex and thrombotic events (Table 1). Similarly, PMF patients with mutant CALR showed lower Hb level (P=0.001) than JAK2 V617F. There was no significant difference in sex, age, WBC count, PLT counts or thrombotic events between PMF patients with mutant CALR and JAK2 V617F (Table 1). For different CALR mutations in ET patients, younger age (P=0.020), lower WBC count (P<0.001), and lower Hb level (P=0.002) were observed in CALR L367fs*46 than JAK2 V617F. In addition, ET patients with CALR K385fs*47 showed lower age (P<0.001), lower WBC counts (P<0.001), lower Hb levels (P=0.025) and higher PLT counts (P=0.005) than JAK2 V617F (Table 2). Table 1. Clinical features of essential thrombocythemia and primary myelofibrosis patients with CALR and JAK2 mutation. Table 2. Clinical characteristics of essential thrombocythemia patients with different types of CALR and JAK2 mutation. The overall survival (OS) rates of patients with ET and PMF were analyzed using the Kaplan-Meier curve. Longer OS was observed in ET and PMF patients with mutant CALR, but not wt CALR (P=0.511 and P=0.729, respectively) (Table 1 and Figure 1D). According to the risk stratification system in ET,11 there was a significant difference between patients with ET in the CALR-mutated group and JAK2 V617F mutant group or wt CALR group (both P<0.001) (Table 1). In summary, our data from this large cohort of Chinese patients with MPNs confirmed CALR mutations were novel molecular markers in wt JAK2 MPNs. It should always be noted that the combination of CALR, JAK2, and MPL W515L/K mutation analysis could contribute to the diagnosis of MPNs.5–7 Different CALR mutations of patients with MPNs had distinct clinical characteristics. Patients with the L367fs*46 and K385fs*47 mutations have shown a favorable prognosis, but further research is required to confirm this result. Given the relative proportion of MPN patients without JAK2/MPL/CALR mutations in our patient group, further investigation should be carried out to find novel molecular aberrations.

Expression of costimulatory molecule B7-H3 and its prognostic implications in human acute leukemia

Abstract Objectives This study focused on the expression pattern and clinical significance of B7-H3 expression in human acute leukemia. Methods We systematically analyzed 134 patients with acute myeloid leukemia (101 cases) and acute lymphocytic leukemia (33 cases) by flow cytometry. Results The frequency of B7-H3+ cases was 44.8% in total. The B7-H3 expression rate differed from 0% to 74.8% in individual cases. The correlation between B7-H3 expression and traditional prognostic factors, such as age and gender, the white blood cell count was not confirmed. However, B7-H3 had a significant higher expression in CD34+ cases and high risk karyotypes. Conclusions Owing to the expression of B7-H3 being statistically relevant in predicting disease progression and a shorter life survival, our results demonstrated that B7-H3 expression in acute leukemia predicts an unfavorable outcome.

The investigation of mutation and single nucleotide polymorphism of receptor tyrosine kinases and downstream scaffold molecules in acute myeloid leukemia

We investigate the role of mutations of receptor tyrosine kinases as well as their downstream scaffold molecules in leukemogenesis of acute myeloid leukemia (AML) in Chinese patients. Genes of interest included FLT3, PDGFRβ, KDR, CSF2Rβ, SOCS1, PIAS3 and SHIP. The coding sequence of these genes was analysed by the reverse transcription-polymerase chain reaction to search novel mutations. A novel mutation (A > T, Q1154L) of SHIP (1 of 192, 0.52%) was identified and another novel mutation (C > T, R685C) of PDGFRβ (2 of 192, 1.04%). In addition, FLT3 mutations were seen in three of five patients with AML following myelodysplastic syndrome (60%) and 39 of 268 (14.6%) de novo AML patients (P < 0.05). No mutations were found in the coding sequence regions of KDR, CSF2Rβ, SOCS1 or PIAS3.

Identification of the STAT5B‐RARα fusion transcript in an acute promyelocytic leukemia patient without FLT3, NPM1, c‐Kit and C/EBPα mutation

T(15;17) is the most common chromosomal aberration in patients with acute promyelocytic leukemia (APL), leading to the formation of PML‐RARα fusion gene. In a small subset of patients with APL, the RARα gene is fused with different partners. Here, we report a rare APL case with STAT5B‐RARα fusion transcript. Cytomorphologic and immunophenotypic analyses showed typical features of APL. However, cytogenetic analysis showed normal karyotype, and interphase fluorescence in situ hybridization (FISH) showed PML‐RARα negative. Quantitative RT‐PCR also showed PML‐RARα negative but STAT5B‐RARα positive and sequencing analysis confirmed the result. Molecular markers including FLT3, NPM1, c‐Kit and C/EBPα mutation were all negative. To our knowledge, this is the first APL patient with STAT5B‐RARα in Chinese population and the fifth patient around the world according to published paper.

Clinical significance of CSF3R , SRSF2 and SETBP1 mutations in chronic neutrophilic leukemia and chronic myelomonocytic leukemia

Chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) are rare hematologic neoplasms. We performed CSF3R, SRSF2 and SETBP1 mutational analyses in 10 CNL and 56 CMML patients. In this sample cohort, 80% of CNL patients harbored CSF3R mutations, of which the CSF3R T618I mutation was dominant. Mutations in CSF3R and SETBP1 were found in 7.1% and 5.3% CMML patients respectively, while 25% of CMML patients carried SRSF2 mutations. Strikingly, we identified that all of the CSF3R mutations detected in CMML patients were represented by a P733T mutation. The CSF3R P733T mutation represents a novel CSF3R mutation. In addition, none of the four CSF3R P733T mutated patients carried SRSF2 mutations [0/14 (0%) patients with combined CSF3R P733T and SRSF2 mutations vs. 4/42 (9.5%) with CSF3R P733T and wt SRSF2, P < 0.001]. Both mut SRSF2 and mut SETBP1 patients had shorter overall survival (OS) and progression-free survival (PFS) compared to patients with wt SRSF2 (P < 0.001 both) and wt SETBP1 (P < 0.001 and P = 0.02, respectively). While we found no significant differences in OS and PFS as a consequence of CSF3R mutation status, our work suggest that the CSF3R T618I mutation is a diagnostic marker with good specificity and sensitivity for CNL. In conclusion, our study highlights effective diagnostic and prognostic markers of CNL and CMML patients in the Chinese population.

Heterogeneous leukemic clones identified by NPM1 mutation analysis in patient with acute monocytic leukemia

Abstract NPM1 mutation is the most common molecular abnormality in patients with acute myeloid leukemia (AML), especially normal karyotype AML (NK-AML), and is associated with a favorable prognosis in the absence of concomitant FLT3-ITD. Like other molecular abnormalities such as FLT3-ITD, C/EBPα and c-Kit mutation, NPM1 mutation normally presents as a recurrent molecular abnormality. The NPM1 mutation is generally used as a molecular marker in the prognosis evaluation of a patient with AML. Here, we report a different case. He was first diagnosed with NPM1 mutation-positive acute monocytic leukemia. However, he achieved no remission, but the NPM1 mutation dramatically became negative after induction chemotherapy. Finally, he achieved complete remission after salvage chemotherapy and the NPM1 mutation was still negative. To our knowledge, this is a rare case according to the worldwide published literature.

The significance of 18F-FDG PET/CT in secondary hemophagocytic lymphohistiocytosis

This study was aimed to investigate the significance of 18F-FDG PET/CT in secondary hemophagocytic lymphohistiocytosis (sHLH) patients. A total of 18 patients received 18F-FDG PET/CT scan at initial diagnosis. All patients (18/18) had at least 3 organs involved, with increased FDG metabolism in different degrees. Fifteen cases (15/18) had definite underlying diseases, including infections (IAHLH), rheumatosis (RAHLH), or malignancy (MAHLH). The SUVmax of patients in MAHLH group was significantly higher than patients in IAHLH group or RAHLH group (P = 0.015, P = 0.045). Furthermore, the SUVmax of patients in IAHLH group was significantly higher than patients of RAHLH group (P = 0.043). Therefore, we concluded that 18F-FDG PET/CT may especially play important role in differential diagnosis of sHLH.

JAK2 V617F patients with essential thrombocythemia present with clinical features of polycythemia vera

Recent studies have shown that Janus tyrosine kinase 2 (JAK2) V617F mutation is found in nearly all patients with polycythemia vera (PV) and underlie the basis of PV molecular pathogenesis. Moreover, JAK2 V617F patients with essential thrombocythemia (ET) have been found to have some clinical features similar to PV. To determine whether the same is true in a different Chinese patient population, we employed Allele-specific polymerase chain reaction in combination with sequence analysis to investigate the point mutation in a series of Chinese patients with hematological malignancies. A total of 99 Chinese myeloproliferative disorder patients and 120 additional patients with acute myeloid leukemia, acute lymphoblastic leukemia and myelodysplastic syndromes were studied. The V617F mutation was detected in genomic DNA of peripheral blood samples of 16 of 23 PV patients (69.6%), 21 of 45 ET patients (46.7%) and 3 of 8 patients with idiopathic myelofibrosis (37.5%). There were striking differences in clinical features such as hemoglobin, hematocrit and neutrophils percentages between V617F positive and negative patients with ET. Hence, our data support the idea that JAK2 V617F mutation divides ET patients into two subtypes, with the V617F positive group showing phenotypic similar to that of PV.

The role of aberrant transcription factor in the progression of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease with distinct biological and clinical features. The Philadelphia (Ph) chromosome and BCR/ABL fusion gene is the major cytogenetic and molecular marker of CML chronic phase. However, the molecular mechanisms of disease progression are not well known. Recent studies have shown that a several of transcription factors such as AML1, C/EBPα, HOX and GATA family play important role in hematopoiesis. In addition, the aberrations of these transcription factors including mutations or translocations have also been found to contribute the disease progression. Hence, BCR/ABL and aberrant transcription factors may result in the phenotype of CML blast crisis together.