Hairong Qiu

Cytogenetic characterisation in Chinese patients with chronic lymphocytic leukemia: A prospective, multicenter study on 143 cases analysed with interphase fluorescence in situ hybridisation

Chronic lymphocytic leukemia (CLL) is infrequent in Chinese people. Conventional cytogenetic analysis underestimates the frequency of chromosome aberrations in CLL due to the low rate of spontaneous mitoses. The aim of this study was to prospectively explore the frequency of chromosomal abnormalities in Chinese patients with CLL using interphase fluorescence in situ hybridisation (FISH) and probes for 12 centromere, 13q14, 14q32, 17p13, 11q22 and 6q23 on 143 patients with CLL. Molecular cytogenetic aberrations were found in 104 patients (72.7%) and 40 patients (28.0%) with more than two abnormalities. The most frequent abnormality was del(13q14) (47.6%), followed by trisomy 12 (21.7%), 14q32 translocation (19.6%), del(17p13) (12.6%), del(11q22) (11.9%) and del(6q23) (4.9%), respectively. The percentages of patients with aberrations by FISH were 75.4%, 72.3% and 67.7% for Binet stages A, B and C, respectively. In early stage (Binet A), del(13q14) aberration was more frequent than in Binet B and C (61.5% vs. 31.9% and 41.9%) (P = 0.021). Patients with advanced stage (Binet C) had more frequent del(17p13) aberration than in Binet A and B (32.3% vs. 9.2% and 4.3%) (P = 0.008). It was showed that the frequencies of the chromosomal abnormalities in our study population were similar to the frequencies in Western countries.

The investigation of JAK2 mutation in Chinese myeloproliferative diseases‐identification of a novel C616Y point mutation in a PV patient

Sir, myeloproliferative diseases (MPD) are a spectrum of pathogenetically related disorders of varying clinical manifestations, characterized by neoplastic expansion of relatively mature granulocyte, erythroid, megakaryocyte, or monocyte and eosinocyte lineage cells. Except the Philadelphia chromosome and BCR-ABL fusion gene as the hallmark genetic abnormality of chronic myelogenous leukemia (CML), the molecular mechanism of most MPD such as PV, ET as well as IMF was not identified. However, a novel point mutation affecting the Janus tyrosine kinase 2 (JAK2 V617F) was described in early 2005 and confirmed by multiple competing groups (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; Levine et al., 2005; Zhao et al., 2005). It has been regarded as novel molecular diagnostic marker of BCR/ABLnegative MPD. To investigate its prevalence and clinical significance in Chinese MPD patients, we introduced Allele-Specific PCR (AS-PCR) in combination with sequence analysis to screen JAK2 V617F mutation. A total of 98 Chinese MPD patients and 120 additional haematological malignancies including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), MDS were analysed for JAK2 V617F mutation. Ninety-eight MPD patients were referred for polycythemia (PV) (n 1⁄4 57), essential thrombocythemia (ET) (n 1⁄4 18), idopathic myelofibrosis (IMF) (n 1⁄4 12), hypereosinophilic syndrome (HES) (n 1⁄4 2) and CML (n 1⁄4 9). A total amount of 2 ml peripheral blood samples of MPD and 5–10 ml bone marrow samples of AML, ALL, MDS at the time of initial diagnosis were obtained with informed consent and genomic DNA was isolated with the use of the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany). In addition, peripheral blood samples from 20 healthy donors were also collected as control. The first forward primer (50-agcatttggttttaaattatggagtatatt-30) of AS-PCR is specific for the mutant allele and contains an intentional mismatch at the third nucleotide from the 30 end. The outer primer pair (forward primer: 50-atctatagtcatgctgaaagtaggagaaag-30; reverse primer: 50-ctgaatagtcctacagtgttttcagtttca-30) was designed to amplify fragments from both mutant and wild-type alleles as internal control. The electrophoresis showed that mutant allele is two bands including 203 and 364 bp but wild-type only 364 bp. The positive samples were all amplified only with the outer primer pair again and confirmed by sequence analysis using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer BioSystems, Foster City, CA, USA) on an ABI Prism 3700 DNA analyser (Perkin-Elmer Bio-Systems). Through AS-PCR combined with sequence analysis, we identified 55 BCR/ABL-negative MPD patients harbouring JAK2 V617F mutation. None of the AML, ALL, MDS, CML was found harbouring JAK2 V617F. The mutation was detected in genomic DNA of peripheral blood samples of 43 of 57 PV patients (75.4%), seven of 18 ET patients (38.9%) and five of 12 IMF patients (41.7%). Furthermore, the mutation was not detected in the HES patient and 20 healthy controls. There is to be mentioned that a novel point mutation-C616Y was identified in a PV patient with V617F mutation. The point mutation was never reported by other group. To our knowledge, this is the first study related to JAK2 mutation in a large cohort of Chinese MPD patients. Through AS-PCR in combination with sequence analysis, we detected JAK2 V617F mutation in BCR/ ABL-negative MPD patients except for AML, ALL, MDS as well as CML. The diagnosis of PV in our patients was made using the following diagnostic criteria: (i) haemoglobin >185 g/l in males or >165 g/l in females. (ii) No elevation of serum erythropoietin. (iii) Palpable or radiological splenomegaly. (iv) Platelet count >400 · 10/l or white blood cells >12 · 10/l. For ET patients the diagnostic criteria were: (i) platelet count >600 · 10/l for at least 2 months. (ii) Enlarged and typical clustering megakaryocytes indicated by bone marrow biopsy. (iii) No evidence of reactive thrombocytosis. The diagnostic criteria for IMF were: bone marrow collagen fibrosis, progressive splenomegaly as well as anaemia (Campbell & Green, 2005). In our study, the incidence of JAK2 V617F mutation was similar to other publications. In addition, we identified a novel point mutation-C616Y. It is interesting that INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Interphase fluorescence in situ hybridization detection of cytogenetic abnormalities in B-cell chronic lymphocytic leukemia.

The most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q14 and 17p13, trisomy 12, and 14q32 rearrangement. Conventional cytogenetic analysis underestimates the frequency of specific chromosome aberrations in B-CLL because of the low rate of spontaneous mitoses and the poor response to mitogen stimulation. We used interphase fluorescence in situ hybridization (I-FISH) to explore the incidence of chromosomal changes in the peripheral blood cells of B-CLL patients. Probes for 13q14 (D13S319), 17p13 (p53), the centromere of chromosome 12 (CEP12), and 14q32 (IGHC/IGHV) were applied to detect chromosomal aberrations in peripheral blood samples from 83 B-CLL patients (60 men, 23 women). Molecular cytogenetic aberrations were found in 61 cases (73.5%), and 8 patients (9.6%) showed 2 kinds of abnormalities. The most frequent abnormality was deletion of 13q14 (41.0%), followed by +12 (19.3%), deletion of 17p13 (12%), and 14q32 rearrangement (9.6%). FISH results were analyzed for correlation with Binet stages. The percentages of patients who showed abnormalities by FISH were 73.0%, 73.3%, and 80% for Binet stages A, B, and C, respectively, and the percentages of patients with abnormalities who showed 2 anomalies were 7.9%, 27.3%, and 0% for Binet stages A, B, and C, respectively. We noted no consistent pattern among the various Binet stages in the distribution of either the types of FISH-detected anomalies or the numbers of FISH anomalies. I-FISH was found to be a rapid, exact, and sensitive technique for analysis of chromosome aberrations in CLL. FISH could provide accurate information regarding the molecular cytogenetic features of CLL.

Intermediate prognosis of 6q deletion in chronic lymphocytic leukemia

Cytogenetic features have an important role in the definition of distinct disease subsets in CLL. The deletion of 6q is known to occur at a relatively low frequency in CLL, and the detailed analysis of hematologic and clinical features of patients with CLL with 6q deletion is limited. To verify the incidence and prognostic significance of 6q deletion in Chinese patients with CLL, fluorescence in situ hybridization (FISH) was used in 240 patients with CLL. del(6q23) was found in 18 patients (7.5%), and only five patients had deletion in 6q23 as the sole abnormality. Strong correlations between del(6q23) and clinical parameters were not found. A difference in terms of survival in patients with del(6q23) as compared with patients without this anomaly was not able to be demonstrated. However, a significant difference was found when comparing the del(6q23) group with the del(17p13) or del(11q22.3) group (p = 0.023), or isolated del(13q14) group (p = 0.019). Our findings place the del(6q23) cytogenetic subset of CLL in an intermediate prognosis position between patients with del(11q22.3) or del(17p13), and patients with isolated del(13q14). FISH probes to detect deletions of 6q might be useful in clinical practice in the work-up of patients with CLL.

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.

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.

Prognostic impact of Epstein-Barr virus (EBV)-DNA copy number at diagnosis in chronic lymphocytic leukemia

Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems.

The analysis of JAK2 and MPL mutations and JAK2 single nucleotide polymorphisms in MPN patients by MassARRAY assay.

Recent studies have shown that JAK2 V617F, MPL W515L/K and JAK2 exon 12 mutations underlie the major molecular pathogenesis of myeloproliferative disorders (MPN). Allele‐Specific Polymerase Chain Reaction (AS‐PCR), direct sequencing and MassARRAY assay were used to ascertain the real prevalence of these mutations and the influence of genetic susceptibility in Chinese MPN patients. The positive rate of JAK2 V617F in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) was 82.0%, 36.6% and 51.1% respectively. One ET patient and two PMF patients harboured the MPL W515L mutation and three PV patients harboured JAK2 exon 12 mutations. All of these patients were confirmed as JAK2 V617F negative. Clinical data demonstrated that PV patients with JAK2 exon 12 mutations were younger, had higher haemoglobin levels and white blood cell counts than PV patients with JAK2 V617F. In addition, through analysis of 4 polymorphic loci of JAK2 gene, no significant difference of distribution frequency was found among PV, ET and PMF patients. Distribution frequency of haplotype also was not significantly different among PV, ET and PMF patients. We conclude that JAK2 V617F is a major molecular pathogenesis in Chinese MPN patients. MPL W515L mutation and JAK2 exon 12 mutations can also be found in JAK2 V617F negative MPN patients.

The Negative Prognostic Impact of Derivative 9 Deletions in Patients Who Received Hydroxyurea Treatment for Chronic Myelogenous Leukemia in the Chronic Phase

Background: Chronic myeloid leukemia (CML) is characterized by the formation of the BCR/ABL fusion gene, as a consequence of the Philadelphia (Ph) translocation between chromosomes 9 and 22. This study was to investigate the incidence and prognostic significance of derivative chromosome 9 (der(9)) deletions in CML patients who received hydroxyurea treatment in the chronic phase. Patients and Methods: Fluorescence in situ hybridization (FISH) analysis was used to assess the der(9) deletion status of 48 CML patients in blast crisis (CMLBC). Results: Among the 48 CML patients, 8 (16.7%) showed der(9) deletions, and the deletions were also existent at diagnosis. The median duration of the chronic phase for patients with der(9) deletions was 18 (range 4–38) months compared to 48 (range 0–204) months for patients without deletions (p < 0.001). Der(9) deletions were not associated with increased karyotypic instability. There was no difference in the probability of the der(9) deletions between the cases having progressed to myeloid or lymphoid blast crisis. Conclusion: The results indicated that the FISH technique could effectively detect der(9) deletions. CML patients with der(9) deletions show more rapid progression and poorer prognosis, and the deletion status is a powerful prognostic factor.

Acute myeloid leukemia in four patients with t(8;21) treated with all-trans retinoic acid as a single agent

Acute myeloid leukemia (AML) is characterized by a block of terminal differentiation of hematopoietic precursors and the repression of normal hematopoiesis by expanding immature blasts. AMLs are frequently associated with specific chromosomal translocations, resulting in the generation of chimeric genes. These genes encode chimeric transcription factors, which possess chromatin-modeling activity and deregulate the transcription of specific target genes[1]. The use of all-trans retinoic acid (ATRA) has significantly improved the treatment results in acute promyelocytic leukemia (APL). The great majority of patients treated with ATRA as a single agent will experience a relapse within a few months. Nevertheless, when used in combination with conventional chemotherapy, the efficacy of ATRA increases dramatically, improving the long-term survival of APL patients to 75% [2]. In non-APL AML, the effects of ATRA are less clear. The majority of nonAPL AML blast cells are sensitive to ATRA in vitro, but a considerable proportion of the cells are resistant [3]. This stresses the need for more knowledge about the molecular mechanisms of ARTA and the demand for tools to predict ARTA sensitivity. A distinct subset of AML, making up *10% of all AML cases, is characterized by a t(8;21)(q22;q22) translocation and, morphologically, by the presence of myeloblasts, showing maturation in the neutrophil lineage. The t(8;21) juxtaposes the RUNX1 gene on chromosome 21 with the CBFT1 gene on chromosome 8, generating the AML1-ETO fusion transcription factor. We now present four patients with AML treated with ATRA alone, who were initially misdiagnosed as APL according to the FrenchAmerican-British (FAB) classification. Case 1: In July 2007, a 25-year-old man with a fever and cough was admitted to other hospital. Peripheral blood counts showed hemoglobin (Hb) 65 g/L, white blood cells (WBC) 23.3610/L, blood platelet cells (plts) 69610/L. Bone marrow examination revealed a hypercellularity with 16% myeloblasts and 52% abnormal promyelocytes in the existence of Auer rods, large granules packed in cytoplasm and nuclei irregular in size and shape. Diagnosis was made as APL based on FAB classification. The patient received arsenic trioxide (10 mg/day) for 30 days, WBC was 15.8610/L with normal platelet count, and the level of promyelocytes in bone marrow was reduced to 18.4%. Then, he was admitted to our hospital and continued to receive arsenic trioxide. At this point, the diagnosis was changed from APL to t(8;21) AML according to karyotype analysis, flow cytometric immunophenotyping of bone marrow cells and reverse transcription polymerase chain reaction (RT-PCR) (Table I). Interphase fluorescence in situ hybridization (FISH) showed 96.7% of AML1-ETO fusion gene in bone marrow cells but failed to demonstrate the presence of PML-RARa fusion gene. Myeloblasts in peripheral blood disappeared, and promyelocytes decreased from 18.4% to 9.16% by the 50th day after arsenic trioxide treatment was initiated. However, FISH analysis for AML1-ETO fusion gene showed a level of 92% in bone marrow cells. The patient received ATRA (40 mg/day) instead of arsenic trioxide. A