Qinrong Wang

EZH2 mutations are related to low blast percentage in bone marrow and -7/del(7q) in de novo acute myeloid leukemia.

The purpose of the present work was to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo acute myelogenous leukemia by sequencing the entire coding region. EZH2 mutations were identified in 13/714 (1.8%) of AML patients were found to be more common in males (P = 0.033). The presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P<0.0001) and -7/del(7q) (P = 0.025). There were no differences in the incidence of mutation in 13 genes, ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. No difference in complete remission, event-free survival, or overall survival was observed between patients with and without EZH2 mutation (P>0.05). Overall, these results showed EZH2 mutation in de novo acute myeloid leukemia as a recurrent genetic abnormality to be associated with lower blast percentage in BM and -7/del(7q).

A BCR - JAK2 fusion gene from ins(22;9)(q11;p13p24) in a patient with atypical chronic myeloid leukemia

In the 2008 World Health Organization (WHO) classifi cation system, myeloproliferative neoplasms (MPNs) are subclassifi ed into eight separate entities: chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofi brosis (PMF), systemic mastocytosis, chronic eosinophilic leukemia not otherwise specifi ed, chronic neutrophilic leukemia and unclassifi able MPN. Th e diagnosis of CML requires the presence of BCR – ABL, and its absence is required for all other MPNs. Moreover, JAK2 V617F has also served as a clonal marker found in most patients with PV, ET or PMF [1].

ETV6 mutation in a cohort of 970 patients with hematologic malignancies

The ETV6 gene (previously known as TEL) belongs to the ETS (E26 transformation specific) family of transcription factors characterized by 2 important domains: the C-terminal Ets domain responsible for specific DNA-binding activities and the N-terminal helix–loop–helix (HLH) oligomerization

Absence of BRAF V600E mutation in hematologic malignancies excluding hairy-cell leukemia

Th e BRAF gene is a member of the Raf kinase family and encodes a serine/threonine-specifi c protein kinase that plays an important role in regulating the mitogen activated protein (MAP) kinase/ERK signaling pathway and aff ects cell division, diff erentiation and secretion. Somatic mutations in the BRAF gene occur in a wide variety of human tumors, most commonly in melanoma (43%) and thyroid (39%), colon (12%) and ovarian (12%) cancers. BRAF missense mutations are mainly localized in the glycine-rich loop and activation segment, encoded by exon 11 and exon 15, respectively. A missense mutation in DNA (1799T → A) resulting in an amino acid change from valine to glutamic acid at codon 600 (V600E) accounts for approximately 80% of BRAF mutations found in human cancers [1]. Recently, the BRAF V600E mutation was found in all 48 patients with hairy-cell leukemia (HCL) in one study [2]. Nevertheless, the BRAF V600E mutation has been reported to be rare in other myeloid and lymphoid neoplasms [2–15]. In order to confi rm the prevalence of the BRAF V600E mutation in hematologic malignancies other than HCL, we investigated the prevalence of this gene mutation in patients with a variety of hematologic malignancies. A total of 780 patients were enrolled in this study, including 288 patients with de novo acute myelogenous leukemia (AML), 84 with de novo myelodysplastic syndromes (MDS), 84 with myeloproliferative neoplasms (MPN), 71 with chronic myeloid leukemia (CML) in chronic phase, 51 with CML in blast crisis, 10 pediatric and 102 adult patients with precursor B-cell acute lymphoblastic leukemia (B-ALL), four pediatric and 13 adult patients with T-cell acute lymphoblastic leukemia (T-ALL), 47 with chronic lymphocytic leukemia (CLL) and 26 with multiple myeloma (MM) with bone marrow infi ltration ( 30%). Diagnoses in these patients were made according to the French–American–British (FAB) classifi cation and revised with application of the World Health Organization (WHO) 2008 classifi cation. Th e disease burden for all patients was above the sensitivity of the Sanger direct DNA sequencing technique. Th e median age of the case series was 42 years (range 5–88 years), and the majority were male (54%) and mostly ethnic (Han) Chinese. Th e main characteristics of patients in this study are summarized in Supplementary Table I to be found online at http:// informahealthcare.com/doi/abs/10.3109/10428194.2012. 695777. Th e study was approved by the Ethics Committee of the First Affi liated Hospital of Soochow University and was conducted according to the Declaration of Helsinki. We examined the BRAF V600E mutation by polymerase chain reaction (PCR) amplifi cation of exon 15 of the BRAF gene followed by direct bidirectional DNA sequencing in cells from 780 patients with hematologic malignancies. Genomic DNA was extracted from frozen bone marrow mononuclear cells (BMMCs) collected at diagnosis after Ficoll gradient centrifugation using standard procedures. For PCR amplifi cation of exon 15 of the BRAF gene, primers 5 ’ -TCATAATGCTTGCTCTGATAGGA-3 ’ (forward) and 5 ’ -GGCCAAAAATTTAATCAGTGGA-3 ’ (reverse) were used for both PCR and sequencing. PCR products were directly sequenced on both strands using an ABI 3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA). Th e aim of this study was to determine the occurrence of the somatic BRAF V600E mutation in patients with hematologic malignancies, excluding HCL, especially in patients with myeloid malignancies, ALL or MM. We sequenced exon 15 in all 780 patients enrolled in the study. However, no evidence of BRAF mutations invoving exon 15, including the most common mutation BRAF V600E, was found in these patients. By contrast, the BRAF V600E mutation was detected in BMMCs from fi ve patients with HCL. Our data confi rm that the BRAF V600E mutation is common in HCL but indicate it to be absent in MDS, AML, MPN, precursor B-ALL, T-ALL, MM and lymphoma, suggesting that it may not be widespread in hematologic malignancies, excluding HCL.

T-SPOT.TB for Detection of Tuberculosis Infection among Hematological Malignancy Patients and Hematopoietic Stem Cell Transplant Recipients

The diagnosis of latent Mycobacterium tuberculosis infection (LTBI) is recommended in hematological malignancy patients and before hematopoietic stem cell transplantation (Guidelines for the prevention and management of infectious complications of solid organ transplantation, 2004). Compared to traditional methods such as tuberculin skin test (TST), T-SPOT.TB has been shown to be more specific. In the present study we enrolled 536 patients for whom T-SPOT.TB was performed, among which 295 patients also received the TST test. The agreement (79%) between T-SPOT.TB and TST was poor (?=0.274, P<0.001). The patients with positive T-SPOT.TB results numbered 62 (11.6%), in which only 20 (48.8%) of the 41 receiving the TST test had positive results. A majority of the patients with T-SPOT.TB positive results had some other evidence ofTB, such as TB history, clinical symptoms and an abnormal chest CT scan. Active TB was found in 9 patients, in which 2 had negative TST results. We followed up the patients and no one developed active TB. Our study suggested that the T-SPOT.TB may be more useful for screening LTBI and active TB in hematological malignancy patients and hematopoietic stem cell transplant recipients than the TST test.

High frequency of BTG1 deletions in patients with BCR-ABL1–positive acute leukemia

Deletions affecting the B-cell translocation gene 1 (BTG1) have recently been reported in 9% of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and occur even more frequently in ETV6-RUNX1-positive and BCR-ABL1-positive subgroups. To investigate whether the BTG1 deletions occur in other BCR-ABL1-positive acute leukemias besides BCP-ALL, we analyzed 44 leukemia cases harboring the BCR-ABL1 transcript [32 BCP-ALL, six mixed-phenotype acute leukemia (MPAL), and six chronic myeloid leukemia in B-lineage blast crisis (CML-BC)] by array-based comparative genomic hybridization and reverse transcription-PCR. BTG1 deletions were present in 31.8% of BCR-ABL1-positive acute leukemia patients, including 31.3% of BCP-ALL (10/32), 33.3% of MPAL (2/6), and 33.3% of CML-BC (B-lineage) (2/6) patients. Of note, the intragenic deletion breakpoints, mapping to 5 different positions at the proximal end of the breakpoint, clustered tightly within exon 2 of BTG1, which were located within a stretch of 20 bp from nucleotide 284 to nucleotide 304 and led to truncated BTG1 transcripts. There were no significant differences in the median white blood cell count, hemoglobin concentration, platelet count, bone marrow blast count, sex, age, or overall complete remission rate between patients with and without BTG1 deletions. Taken together, our data suggest that BTG1 deletions might play a role in leukemogenesis of BCP-ALL as well as of BCR-ABL1-positive MPAL and CML-BC (B-lineage).

Transcriptome sequencing reveals CHD1 as a novel fusion partner of RUNX1 in acute myeloid leukemia with t(5;21)(q21;q22)

BackgroundRUNX1/AML1, which is a Runt family transcription factor critical for normal hematopoiesis, is frequently mutated or translocated in a broad spectrum of hematopoietic malignancies.FindingsWe describe here the case of a 54-year-old female developed acute myeloid leukemia with a t(5;21)(q21;q22). Transcriptome sequencing identified the chromodomain-helicase-DNA-binding protein 1 gene, CHD1, as a novel partner gene of RUNX1. Furthermore, the patient was found to harbor FLT3-ITD mutation, which might collaborated with CHD1-RUNX1 in the development of acute myeloid leukemia.ConclusionsWe have identified CHD1 as the RUNX1 fusion partner in acute myeloid leukemia with t(5;21)(q21;q22).

Somatic mutations of PHF6 in patients with chronic myeloid leukemia in blast crisis.

∗ Denotes equal contribution. Correspondence: Dr. Suning Chen, Jiangsu Institute of Hematology, Key Laboratory of Thrombosis and Hemostasis of Ministry of Health, the First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou 215006, P. R. China. Tel: 086-0512-67780441; Fax: 0086-512-65113556. E-mail: chensuning@sina.com (or) Dr. Yun Zhao, Cyrus Tang Hematology Center, Soochow University, 199 Ren ’ ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, P. R. China 215123. Tel: 86-512-65880899, ext. 501; Fax: 86-512-65880929. E-mail: zhaoy@suda.edu.cn

Immunophenotype of Chinese Patients with T-Lineage Acute Lymphoblastic Leukemia and Its Association to Biological and Clinical Features

Background/Aims: To investigate the immunophenotype of 46 Chinese patients with T-lineage acute lymphoblastic leukemia (T-ALL) and its association with biological and clinical features. Methods: 46 patients with T-ALL were immunophenotyped by flow cytometry, and 30 cases were also subjected to karyotype analysis by R-banding technology. The clinical and biological characteristics of T-ALL patients between MyAg+ and MyAg– groups were analyzed. Results: Myeloid antigen (MyAg) expression was documented in 41.3% of the 46 T-ALL cases. Abnormal karyotypes were detected in 17 out of 30 (56.7%) cases. Our data showed that the median lowest white blood cell (WBC) count and lowest hemoglobin level, higher CD34 positivity, and a lower proportion of patients with splenomegaly were found to be correlated with MyAg+ T-ALL. No statistical difference was noted in the complete remission (CR) rate, relapse rate, induction death rate or total death rate among MyAg+ and MyAg– patients. In our cohort, none of the antigens tested affected the CR rate after the first induction. Conclusion: Our results indicate that MyAg expression in patients with T-ALL was not associated with adverse presenting clinical and biological features as well as response to induction treatment. The expression of surface antigens had no impact on initial chemotherapy CR achievement.

ASXL1 mutations are frequent in de novo AML with trisomy 8 and confer an unfavorable prognosis

Acute myeloid leukemia (AML) is a heterogeneous group of genetically diverse hematopoietic malignancies with variable response to treatment.[1] Among the clinical and laboratory characteristics which influence the response to treatment and the survival of patients, cytogenetics at diagnosis represents the most important prognostic variable. Trisomy 8 is one of the most frequent recurring numerical chromosome aberrations in AML, occurring at a frequency of 10%.[2] It presents either as the sole cytogenetic abnormality or in combination with a large variety of different cytogenetic abnormalities. Patients with sole trisomy 8 are mostly classified as having an intermediate prognosis.[3,4] Additional sex combs like-1 (ASXL1) gene, encodes a highly conserved protein that belongs to the enhancer of trithorax and polycombgenes, a gene family with dual functions in both epigenetic activation and repression of gene transcription.[5] Frameshift and stop mutations were found that were predicted to lead to loss of the carboxyterminal plant homeodomain finger on the protein level.[6] ASXL1 mutations (ASXL1-mut) have been detected in about 6–30% of AML.[7–10] Recent reports have demonstrated that ASXL1-mut are more frequent in patients with aberrant karyotype especially in cases with trisomy 8.[11,12] These suggested that ASXL1-mut might play an important role in the patients with trisomy 8. However, the roles of ASXL1-mut in these patients are still unclear. Here, we investigated the occurrence and prognostic impact of ASXL1-mut in AML patients with trisomy 8 aberration. A total of 78 patients with trisomy 8 as the sole aberration or accompanied with other anomalies enrolled from 2007 to 2013 in Jiangsu Institute of Hematology were retrospectively analyzed in this study which was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University following Declaration of Helsinki. The median age was 43 years (12–84 years), and the cohort was all Chinese with the majority of male (51.3%). Historically, analyses have divided patients into favorable, intermediate, and unfavorable cytogenetic groups.[13,14] The same classification scheme was used for this analyses. Here, favorable subgroup consisted of 9 cases with trisomy 8 in combination with t(15;17) or t(8;21), the intermediate group comprised of 41 cases with trisomy 8 as the sole aberration or accompanied with one anomaly which did not include the favorable cytogenetic abnormalities, and the unfavorable category included 28 patients with complex (two or more) aberrations. Only patients of de novo AML with trisomy 8 who received intensive treatment were included into the prognostic analyses. Chromosome analyses were performed according to standard protocols using R-banding. The chromosomes were classified according to the International System for Human Cytogenetic Nomenclature (ISCN, 2005). Genomic DNA was prepared from purified fraction of mononuclear cells after Ficoll density centrifugation. Then PCR amplification of the entire coding region of ASXL1 exon 12 was performed followed by direct bidirectional DNA sequencing. The positive mutation samples were analyzed twice to confirm the results. Only frameshift and nonsense mutations of ASXL1 were analyzed in this study. All statistical analyses were performed using SPSS version 17.0 (SPSS Inc., Chicago, IL). Comparisons of continuous and dichotomous variables were based on the Kruskal–Wallis test and chi-square test respectively. Survival curves were calculated for overall survival (OS) and event-free survival (EFS) according to Kaplan–Meier. Comparisons between groups were based on the twosided log-rank test. Statistically significant tests were identified with p value less than 0.05. The frequency of ASXL1-mut was identified in 19.23% (15/78) of AML patients with trisomy 8 as the sole aberration or accompanied with other anomalies by sequencing