Coexistence of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia: A report of 4 cases.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17-25% of patients with acute lymphocytic leukemia and 0.9-3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-ß-myosin heavy chain 11 (CBFß-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFß-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFß-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFß-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFß-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFß-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFß-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

[Autophagy Activity of CD34+ Cells in MDS Patients and Its Clinical Significance].

OBJECTIVE: To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance. METHODS: The activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM). RESULTS: The autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P<0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) . CONCLUSION: The autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.

Andrographolide suppresses thymic stromal lymphopoietin in phorbol myristate acetate/calcium ionophore A23187-activated mast cells and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like mice model.

BACKGROUND: Atopic dermatitis (AD) is one of the most common inflammatory cutaneous diseases. Thymic stromal lymphopoietin (TSLP) has been demonstrated to be an important immunologic factor in the pathogenesis of AD. The production of TSLP can be induced by a high level of intracellular calcium concentration and activation of the receptor-interacting protein 2/caspase-1/NF-κB pathway. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone, has been found to exert anti-inflammatory effects in gastrointestinal inflammatory disorders through suppressing the NF-κB pathway. OBJECTIVE: To explore the effect of ANDRO on the production of TSLP in human mast cells and AD mice model. METHODS: We utilized enzyme-linked immunosorbent assay, real-time reverse transcription polymerase chain reaction analysis, Western blot analysis, and immunofluorescence staining assay to investigate the effects of ANDRO on AD. RESULTS: ANDRO ameliorated the increase in the intracellular calcium, protein, and messenger RNA levels of TSLP induced by phorbol myristate acetate/calcium ionophore A23187, through the blocking of the receptor-interacting protein 2/caspase-1/NF-κB pathway in human mast cell line 1 cells. ANDRO, via oral or local administration, also attenuated clinical symptoms in 2,4-dinitrofluorobenzene-induced AD mice model and suppressed the levels of TSLP in lesional skin. CONCLUSION: Taken together, ANDRO may be a potential therapeutic agent for AD through suppressing the expression of TSLP.

[Clinical and Laboratorial Characteristics of Primary Acute Myeloid leukemia with Philadelphia Chromosome and Inversion 16].

OBJECTIVE: To summarize the clinical characteristics as well as diagnosis and treatment in 1 case of acute myeloid leukemia(AML) with coexpression of Ph and inv(16). METHODS: A series of clinical tests, the cellular morphological, immunological, cytogenetic and molecular biological examinations of leukemia cells were performed. RESULTS: The clinical characteristics of this patient were very common. The cellular morphology is similar to the AML with inv(16). The leukemia cells were stained positively for CD13, CD33, CD34, CD117 and HLA-DR. Karyotypic analysis showed a complex chromosome abnormality including inv(16) and Ph, and the FISH analysis showed that the percentage of rearrangement of CBFß allele was over that of the BCR-ABL fusion signals. The obvious adverse events did not occur in this patient within 3 years. CONCLUSION: Ph as secondary aberration of inv(16) rarely occures in primary AML cases, and so far there have not been the clear criteria of diagnosis and treatment. The cytogenetic and molecular biology could provide the basis for diagnosis. Moreover, autologous hematopoietic stem cell transplantation combined with imatinib probably is one of the effective treatment methods.

Clonal origin and evolution of myelodysplastic syndrome analyzed by dysplastic morphology and fluorescence in situ hybridization.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem/progenitor cells. As bone marrow cells are extremely diverse in these disorders, the origin and evolution of MDS clones are difficult to identify and trace. Cellular dysplasia is a distinct morphologic feature; however, whether the dysplastic cells represent abnormal clones or only nonspecific superficial phenomena remains to be clarified. To address this question, 97 patients were examined for dysplasia features, among them bone marrow slides of 16 patients with chromosomal abnormalities were subjected to fluorescence in situ hybridization (FISH) to determine the karyotype of these dysplastic cells. Furthermore, the emerging frequencies of abnormal karyotypes in various differentiated stages of each lineage were also evaluated by a combination of morphological evaluation and FISH karyotyping. Our results indicate that the overall percentage of dysplastic cells does not differ significantly among the WHO subtypes, while the megakaryoid lineage presents the most frequent dysplasia in all subtypes. A positive correlation between dysplastic cells and FISH-detectable abnormal clones was observed, but the dysplastic morphology was not a specific feature of FISH-detectable abnormal clones. FISH-detectable abnormal clones can differentiate into mature granulocytes and erythrocytes, in coexistence with cells originating from the normal clones.

[Analysis of correlation between serum thymidine kinase 1 and acute myeloid leukemia].

This study was purposed to investigate the clinical significance of serum thymidine kinase 1 (STK1) level change in acute myeloid leukemia (AML). Peripheral blood samples of 60 newly diagnosed AML patients were collected and the STK1 levels were determined by enhanced chemiluminescent dot-blot method before and at two weeks after start of inductive treatment and in consolidatory treatment. Using non-parametric test, the differences between groups were analyzed. Then the correlation between STK1 level and clinical characteristics was explored by a way of chi-square test. The results indicated that the serum TK1 level in complete remission (CR) or partial remission (PR) AML patients decreased in varying degree as compared to pretreatment (P < 0.05), while there was no significant difference of TK1 level in non-remission (NR) ones (P > 0.05). The serum TK1 level in CR patients remained low level but increased noticeably after relapse into progressive disease (P < 0.05). A significant correlation was found between STK1 level and chromosomal abnormalities, serum LDH level as well as whether had fever in de novo AML patients (P < 0.05). It is concluded that the serum TK1 level change may be applied for reflecting the aggressiveness of disease, monitoring the clinical response to chemotherapy, evaluating the prognosis and predicating the relapse risk. The decrease of TK1 level suggests effective treatment and tumor burden reduction, while its increase indicate poor prognosis and relapse risk.

[Clinical and laboratorial analysis for 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or positive BCR-ABL].

The purpose of this study was to summary the clinical and laboratorial features in 15 adult cases of mixed phenotypic acute leukemia with Ph chromosome and/or BCR-ABL fusion gene positive (Ph(+)MPAL), 15 adult patients with Ph(+)MPAL were defined by WHO-2008 classification. The clinical characteristics, results of morphology, immunology, cytogenetics and molecular genetic detections and results of follow-up in 15 adult patients with Ph(+)MPAL were analyzed retrospectively. The results showed that 15 patients among 87 cases of MPAL demonstrated Ph(+)MPAL (17.2%; 15/87) (7 males and 8 females), their median age was 51 (range 16-81) year old and median WBC count at diagnosis was 69 (12.7-921)×10(9)/L. Based on FAB criteria, these patients showed different morphologic types, including AML (13.3%; 2/15), ALL (40.0%; 6/15), HAL (46.7%; 7/15). Immunologic analysis indicated that 15 cases of Ph(-)MPAL were all classified as B-lymphoid +myeloid mixed immunophenotype. Except one patient, all expressed CD34 antigen on the surface of leukemia cells with 64.3% strong positive, only Ph (53.3%; 8/15), Ph with additional chromosomal abnormalities (33.3%; 5/15) and normal karyotype (13.3%; 2/15) were cytogenetically identified. BCR-ABL fusion gene transcript positive were detected by multiplex reverse transcription PCR in all cases, with e1a2 subtype (p190) (40.0%; 6/15) and b2a2 or b3a2 (p210) subtype (60.0%; 9/15). Four out of 7 (57.1%) patients were found to have IKZF1 gene deletion, without other common gene mutations. Seven out of 10 cases (70.0%) achieved complete remission (CR) after one cycle of induction chemotherapy. In the induction stage, CR rate seemed higher when tyrosine kinase inhibitors (TKI) were added to chemotherapy (83.3%:50.0%; P = 0.206). Overall survival (OS) in 4 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) was longer than that in 4 patients received chemotherapy alone (P = 0.004). It is concluded that Ph(+)MPAL mainly is expressed as B+My phenotype. The majority of patients is older and has CD34 overexpression. In the aspect of molecular genetics, the Ph(+)MPAL is similar to other acute leukemia with Ph chromosome. Ph(+)MPAL is a subtype of acute leukemia with poor prognosis. WBC count at diagnosis is an independent prognostic factor. The combination of TKI and allo-HSCT can improve their long-term survival, which needs to be confirmed through carrying out a prospective and multicenter clinical trial for newly diagnosed Ph(+)MPAL.

[Clinical significance of common leukemia gene mutations in patients with acute promyelocytic leukemia].

This study was aimed to explore whether multiple common gene mutations of leukemia synergistically involved in acute promyelocytic leukemia (APL) pathogenesis, and to investigate their relevance to clinical features, cytogenetics and molecular risk stratification. 84 specimens of admitted de novo APL patients from February 2005 to October 2010 were collected, the gene mutations of bone marrow mononuclear cells and clinical features of mutation-positive patients were analyzed by genomic DNA-PCR. The results indicated that the prevalence of mutations was 60.7% (51/84), in which the mutations with the highest incidence were found as FLT3-ITD, reaching 27.4% (23/84). Next, there were 12 cases WT1 mutation, 9 for FLT3-TKD, 7 for TET2, 5 for N-RAS, 4 for ASXL1, 2 for EZH2 mutation and 1 positive case in MLL-PTD, IDH1 and CBL mutation respectively. No mutation was found in other JAK1, DNMT3, c-Kit, NPM1, IDH2, RUNX1 and JAK2 (V617F) common leukemia-related genes. Combined analysis with clinical data demonstrated that the patients with FLT3-ITD mutation displayed higher white blood cell counts, while the patients with N-RAS mutation showed lower platelet counts. Overall survival of these patients was obviously shorten as compared with patients with wild-type. This difference between mutant and wild-type of all above mentioned cases was statistically significant (P < 0.05). The difference between APL with simple t (15;17) and additional abnormal karyotype was not statistically significant. It is concluded that the FLT3-ITD mutation is recurrent genetic change in APL, and together with N-RAS mutation indicates poor prognosis. Additional abnormal karyotype does not associate with prognosis of APL.

Long-term survey of outcome in acute promyelocytic leukemia: a single center experience in 340 patients.

The aims of this study are to investigate the outcome and prognostic factors influencing long-term survival on patients with acute promyelocytic leukemia (APL). A total of 340 APL patients admitted to the Department of Hematology from January 1988 to December 2009 were enrolled in this study. All patients received all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) with anthracycline-based induction therapy. After three courses of consolidation chemotherapy, 279 patients received 2 years of maintenance therapy. Survival analyses were carried out using the Kaplan-Meier method and the Cox regression model. In total, 288 achieved CR with the CR rate of 84.7%, and 50 patients died during induction therapy. Univariate analysis identified the following three risk factors for hemorrhagic mortality: fibrinogen level (<1.0 g/l) (P = 0.0007), initial peripheral WBC count(>4 × 10(9)/l) (P = 0.0001), as well as the presence of coagulopathy(P < 0.0001). With a median follow-up of 49 (6-255) months, the estimated 5-year overall survival (OS) and relapse-free survival (RFS) were (89.0 ± 2.4)% and (83.7 ± 2.6)%, respectively. Cox regression analysis of the 290 patients showed initial WBC count, years of diagnosis, and the status of PML-RARα in remission seemed to be independent prognostic indicators for OS and RFS (P = 0.03, P < 0.01 and P = 0.0001, respectively). Cytogenetics in addition to above three variables remained significant for RFS (P = 0.01). Our retrospective observations suggest that the combination of ATRA and/or ATO with anthracycline-based therapy may have useful implications in the perspective of long-term prognosis for adult APL.

[Analysis of tyrosine kinases gene mutations in core binding factor related acute myeloid leukemia and its clinical significance].

OBJECTIVE: To assess the prevalence of several tyrosine kinases (TKs) gene mutations including c-Kit, FLT3 and JAK2 V617F in core binding factor related acute myeloid leukemia (CBF-AML), and analyze their impact on clinical characteristics and prognosis. METHODS: Mutations of c-Kit, FLT3-ITD and FLT3-TKD were detected by genomic DNA PCR and sequencing, and JAK2 V617F mutation screening by allele-specific PCR in 58 newly diagnosed CBF-AML patients [28 AML with inv(16) and 30 with t(8;21)], and analyze the patients clinical characteristics and prognoses. RESULTS: c-Kit aberrations were detected in 32.8% cases, including 6 cases mutated in exon 8 (mutKIT8) and 13 mutated in exon 17 (mutKIT17). MutKIT8 was more prominent in inv(16) than in t(8;21) patients (21.4% vs 0, P = 0.009). Only 2 cases had FLT3-ITD and 7 (12.1%) FLT3-TKD mutations. The result of JAK2 V617F mutation screenings in these CBF-AML patients was negative. The frequency of receptor tyrosine kinases(RTK) mutations was 46.6% and only one case had two kinds of missense mutations (mutKIT8 & TKD(+)). Median age of onset was higher for mutKIT17 than for wide-type c-Kit (wtKIT) patients (55 vs 31, P = 0.003). c-Kit mutations were significantly associated with decreased overall survival (OS) and continuous complete remission (CCR) rates (P = 0.053, and 0.048 respectively), and so did more for exon17 mutated patients reduced (P = 0.005, and 0.013 respectively). FLT3-TKD mutation showed no effects on prognosis of CBF-AML patients. CONCLUSIONS: RTK mutations are common in patients with CBF-AML. c-Kit mutations frequently and JAK2V617F mutation rarely appear in CBF-AML. c-Kit mutations, especially mutKIT17 confers higher relapse risk and poorer prognosis.

[Clinical and laboratory features of patients with CD34(+) acute promyelocytic leukemia].

OBJECTIVE: To explore the expression of CD34 in patients with acute promyelocytic leukemia (APL) and investigate the clinical and laboratory features of CD34(+) APL patients. METHODS: 262 APL patients diagnosed by chromosome analysis and/or fusion gene examination in the last five years were retrospectively analyzed in this study. To survey the expression of CD34 in those patients, all the cases were divided into two groups (CD34(+) APL vs. CD34(-) APL). The clinical features including age, gender, abnormal values of the peripheral hemogram before treatment, the complete remission (CR) rate and the incidence of DIC and laboratory data such as the results of morphology, immunology, cytogenetics and molecular biology (MICM) between those two groups were compared. RESULTS: Of the 262 APL patients, 38 (14.5%) cases were positive for CD34 expression. There were no statistically significant differences between CD34(+) APL and CD34(-) APL groups in gender and age (P > 0.05). Before treatment, the median level of WBC in CD34(+) APL was 25.92 x 10(9)/L, which was significantly higher than that of CD34(-) APL (5.3 x 10(9)/L, P < 0.05). CD34(+) APL by morphology classification were mostly of the subtypes M3b and M3v (65.8%), while these subtypes in CD34(-) APL (40.3%) were significantly less (P < 0.01). There were no statistically significant differences between the two groups compared in respect of complete remission (CR) rate and the incidence of DIC (P > 0.05). The expression level of CD34 in APL had correlation to the expression level of CD2, CD7 and CD117; the latter three phenotypes in CD34(+) APL were significantly higher than those in CD34(-) APL (P < 0.01). No significant difference was found between those two groups by chromosome analysis, but there was more PML-RAR-alpha transcript short form in CD34(+) APL than that in CD34(-) APL (P < 0.05). CONCLUSION: CD34(+) acute promyelocytic leukemia is a unique subtype of APL with different biological characteristics.

Prognostic analysis of refractory anaemia in adult myelodysplastic syndromes.

BACKGROUND: Patients with myelodysplastic syndrome (MDS) display a very diverse pattern. In this study, we investigated prognostic factors and survival rate in adult patients with MDS refractory anaemia (MDS-RA) diagnosed according to French-American-British classification and evaluated the International Prognostic Scoring System (IPSS) for Chinese patients. METHODS: A multi-center study on diagnosis of MDS-RA was conducted to characterize the clinical features of Chinese MDS patients. The morphological criteria for the diagnosis of MDS-RA were first standardized. Clinical data of 307 MDS-RA patients collected from Shanghai, Suzhou and Beijing from 1995 to 2006 were analyzed using Kaplan-Meier curve, log rank and Cox regression model. RESULTS: The median age of 307 MDS-RA cases was 52 years. The frequency of 2 or 3 lineage cytopenias was 85.6%. Abnormal karyotype occurred in 35.7% of 235 patients. There were 165 cases (70.2%) in the good IPSS cytogenetic subgroup, 44 cases (18.7%) intermediate and 26 cases (11.1%) poor. IPSS showed 20 (8.5%) categorized as low risk, 195 cases (83.0%) as intermediate-I risk and 20 cases (8.5%) as intermediate-II risk. The 1-, 2-, 3-, 4- and 5-year survival rates were 90.8%, 85.7%, 82.9%, 74.9% and 71.2% respectively. Fifteen cases (4.9%) transformed to acute myeloid leukaemia (median time 15.9 months, range 3 - 102 months). Lower white blood cell count (< 1.5 x 10(9)/L), platelet count (< 30 x 10(9)/L) and cytogenetic abnormalities were independent prognostic factors by multivariate analysis, but age (= 65 years), IPSS cytogenetic subgroup and IPSS risk subgroup were not independent prognostic factors associated with survival time. CONCLUSIONS: Chinese patients were younger, and had lower incidence of cytogenetic abnormalities, more severe cytopenias but a more favourable prognosis than Western patients. The major prognostic factors were lower white blood cell count, lower platelet count and fewer abnormal karyotypes. The international prognostic scoring system risk group was not an independent prognostic factor for Chinese myelodysplastic syndrome patients with refractory anaemia patients.

[The clinical and laboratory features of acute promyelocytic leukemia: an analysis of 513 cases].

OBJECTIVE: To investigate the clinical and laboratory features of acute promyelocytic leukemia (APL). METHODS: 513 APL patients in the last two decades were retrospectively analyzed in this research. We investigated the clinical features including age, sex, abnormality of peripheral hemogram before treatment, therapeutic effect and follow-up and laboratory data such as morphology, immunology, cytogenetics and molecular biology (MICM). RESULTS: The median age of the APL patients was 33 years old and the ratio of male and female was 1.21:1. Before treatment, the median level of WBC was 4.3 x 10(9)/L and the detection rate of abnormal promyelocyte on blood film was 85.8%; with immunophenotypic detection, the expression levels of CD117, CD34, HLA-DR, CD7, CD14 and CD19 in APL were found to be lower and the expression levels of CD2, CD33 and MPO higher than those in other subtypes of acute myelocytic leukemia (AML) (both P < 0.01). Specific abnormal chromosome t (15;17) was detected in 91.7% of the patients, of whom 75.9% had standard translocation of t (15;17), being the most common one and 15.8% of the patients had t (15;17) with additional abnormal chromosome. There was only 7.5% of the patients with normal karyotype. However, the presence of both simple translocation and complex translocation was seldom seen. With molecular biological detection, PML/RARalpha fusion gene positive rate was 99.6%. In a relatively long clinical follow-up, we found that the complete remission (CR) rate in APL patients was 84.7%, incidence of DIC was 13.4% and five-year survival rate was 30.7%. The median count of WBC in CR group was lower than that non-remission group (P < 0.01). There were no significant differences on expressions of CD34 and CD2 and changes of cytogenetics between the two groups (P > 0.05). CONCLUSIONS: Comprehensive evaluation of MICM could be of important significance in the diagnosis and prognosis judgment for APL patients. The CR rate in these patients with high WBC count was considerable low.

[Prevalence and clinical significance of FLT3 mutations in acute promyelocytic leukemia].

OBJECTIVE: To evaluate the prevalence of Fms-Like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) of juxtamembrane region and point mutation in the second tyrosine kinase domain (TKD) in acute promyelocytic leukemia (APL) and its clinical significance. METHODS: Bone marrow mononuclear cells from 160 newly diagnosed APL patients were analyzed. Polymerase chain reaction (PCR) was used to detect FLT3-ITD mutations, FLT3-ITD positive samples were further analyzed for the ITD allelic ratio (ITD-AR, mutant-wild type ratio). The FLT3-TKD mutation was analyzed by PCR amplification of exon 20 followed by EcoR V digestion and sequencing. RESULTS: Out of 160 patients, 30 (18.75%) patients were FLT3-ITD positive, 17 (10.62%) were FLT3-TKD positive, 2 had both of mutations. The initial WBC count and the ratio of short type PML-RAR alpha isoforms in FLT3-ITD positive and FLT3-TKD positive patients were all higher than that in patients with wild-type FLT3 (FLT3-wt) (P < 0.05). For FLT3-ITD positive patients, the incidences of retinoic acid syndrome (RAS) and disseminated intravascular coagulation (DIC) were 41.7% and 65.4%, respectively, being higher than that of FLT3-wt patients, while their complete remission (CR) rate was lower (69.2% vs 90.3%, P < 0.05). For FLT3-TKD positive patients, the incidence of RAS, DIC and CR rate were not significantly different from that of FLT3-wt patients (P > 0.05). FLT3-ITD positive patients had a shorter overall survival (OS) (P < 0.05), but not disease-free survival (DFS) (P > 0.05) as compared with FLT3-wt patients. There was no significant difference in either OS or DFS between FLT3-TKD positive and FLT3-wt patients. The ITD-AR of 30 FLT3-ITD positive patients varied from 0.11 to 6.55 with a median of 1.0. The initial WBC count, incidence of RAS and DIC, CR rate were not significantly different between the patients with ITD-AR greater than 1.0 and lower than 1.0 (P > 0.05). CONCLUSIONS: FLT3 mutations (FLT3-ITD or FLT3-TKD) are frequently identified in patients with newly diagnosed APL, both mutations are associated with higher initial WBC and short type PML-RAR alpha isoforms. FLT3-ITD mutation is more frequent than FLT3-TKD mutation, and predicts a poorer prognosis, whereas FLT3-TKD mutation does not show the same unfavorable prognostic effect on APL patients.

[Detection of clonal immunoglobulin and T-cell receptor gene rearrangements in newly diagnosed adult patients with acute lymphoblastic leukemia by using multiplex PCR protocols].

OBJECTIVE: To provide the evidence of RQ-PCR-based assessment of minimal residual disease (MRD), the clonal immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements were identified in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) by multiplex PCR protocols. METHODS: Forty newly diagnosed adult patients with B-lineage (B-) and T cell (T-) ALL were involved in this study. All DNA samples were obtained from the bone marrow (BM) mononuclear cells (MNC). IgH, IgK, TCRB, TCRG and TCRD gene rearrangements were detected by BIOMED-2 multiplex PCR protocols, which included 96 different primers and 14 multiplex PCR tubes. RESULTS: The clonal immunoglobulin (Ig) rearrangements were found in 96% of B-ALL, 86% being IgH and 14% IgK. While in T-ALL, clonal TCR rearrangements were found in all of the patients, 83% being TCRB, 78% TCRG and 39% TCRD. More than two clonal markers were found in 91% of B-ALL and 89% of T-ALL patients. CONCLUSIONS: The detection rate of clonal rearrangements using the BIOMED-2 14 multiplex PCR tubes is high, which can detect virtually all clonal B and T-cell proliferations. It can be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

[Analysis of NPM1 gene mutations in acute myeloid leukemia].

OBJECTIVE: To evaluate the prevalence of nucleophosmin (NPM1) gene exon 12 mutations in adults with acute myeloid leukemia (AML) and its clinical characteristics. METHODS: Genomic DNAs from 101 AML adults were screened by PCR and sequencing or capillary electrophoresis (CE) for NPMI mutations. RESULTS: NPM1 exon 12 mutations were present in 31.7% of the overall cohort, including 1/1 (100%) of M0, 9/17(52.9%) of M1 , 7/25 (28.0%) of M2, 0/23(0%) of M3, 2/7 (28.6%) of M4 and 13/25 (52.0% ) of M5. NPM1 gene mutations were more prevalent in patients with normal karyotype (27/59, 45.8%) compared with that in those with karyotypic abnormalities (5/42, 11.9% ) (P < 0.001). NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count (P < 0.05) and low expression of CD34 (P < 0.05) and CD17 (P<0.05). Sequence analysis of these NPM1 mutant cases revealed 5 known mutations (type A, B, D, N(M), and P(M)) and 1 novel variant (named as type S). CONCLUSIONS: NPM1 exon 12 mutations occur with a considerable percentage in AML patients with normal karyotype, M1/M5 subtype and older age, and are associated with higher peripheral white cell count and lower expression of CD34 and CD117.

[MIC categorization of acute lymphoblastic leukemia with myeloid surface antigen expression].

OBJECTIVE: To explore the characteristics of morphology, immunophenotype and cytogenetics (MIC) of myeloid surface antigen-expressing acute lymphoblastic leukemia (My+ ALL). METHODS: One hundred and twenty untreated acute lymphoblastic leukemia (ALL) patients were diagnosed by standard bone marrow smear morphologic analysis and peroxidase staining. Flow cytometry and myeloid monoclonal antibodies (McAb) were used to analyze immunophenotype. Chromosome karyotypes were analyzed by R-band technique. RESULTS: Of 120 cases, 66 (55%) were My+ ALL, including 50 cases of My+ B-ALL (56.8% of B-ALL ), 14 cases of My T-ALL (50% of T-ALL) and 2 cases of My+ T and B-ALL (50% of T and BALL). Of 66 My+ ALL, 10 cases (15.1%) were misdiagnosed as acute non-lymphoblastic leukemia (ANLL), the other 54 My- ALL cases were correctly diagnosed. The inconsistent rate between morphological and immunophenotype classifications was higher in My+ ALL than in My- ALL , and there were more atypical morphology cases in My+ ALL than in My- ALL (P < 0.01). In My+ ALL cases 95.5% expressed CD13, 81.8% CD33, 77.3% CD13 and CD33 simultaneously, and 1.5% CD117, but none CD14, CD15 and MPO. CD34 expression rate in My+ ALL cases was significantly higher than that in My- ALL (P < 0.01 ). Cytogenetic abnormalities rates in My+ ALL and My- ALL were 72.3% and 66.7% (P > 0.05) respectively. t(9;22) and t(9;22) plus other cytogenetic abnormalities were detected more frequently in My+ LL cases than in My- B-ALL (P < 0. 01), and not in My+ T-ALL and My- T-ALL cases. The complete remission (CR) rates was 83.9% in My+ ALL and 79% in My- ALL(P > 0.05). CONCLUSION: My+ ALL had a specific characteristics in morphology, immunophenotype and cytogenetics. Some cases have a myeloid morphologic appearance and might be misdiagnosed as acute myeloid leukemia (AML). My+ ALL have a higher CD34 expression rate than My- ALL. t(9;22) abnormality was more frequently observed in My B-ALL than in My- B-ALL. There was no significant difference in CR rate between My+ ALL and My- ALL.

[A study of NPM1 and FLT3 gene mutations in acute myeloid leukemia].

OBJECTIVE: To evaluate the prevalence and impact of NPM1 gene mutations and FLT3 internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML). METHODS: Mononuclear cells in bone marrow samples were collected from 86 adult patients with newly diagnosed AML. FLT3 and NPM1 genes were amplified with genomic DNA-PCR. NPM1 exon-12 mutations were evaluated with capillary electrophoresis and FLT3-ITD mutations were determined with agarose electrophoresis. RESULTS: NPM1 gene mutations were identified in 29 of the 86 (33.7%) patients and FLT3-ITD in 15 of the 86 (17.4%) patients. In AML patients with normal karyotype, the frequencies of the two abnormalities were significantly higher than in those without (46.0% and 24.0%, P < 0.05). Both mutations were related to high peripheral white blood cell counts (P < 0.05). WBC counts in the six of the seven cases with NPM1+/FLT3-ITD+ AML were significantly higher (>50 x 10(9)/L). Compared with NPM1(-)AML patients, NPM1(+)AML patients were associated with lower expression of CD(34) (P < 0.001), higher complete remission (CR) rate after initial therapy (66.7% vs 53.3%, P > 0.05) and better overall survival (OS) (P > 0.05), but compared with FLT3-ITD(-)AML patients, FLT3-ITD+ AML patients were associated with lower CR rate (50.0% vs 58.8%; P > 0.05) and worse OS (P > 0.05). The CR rates were 66.7%, 62.5%, 50.0% and 42.9%, respectively, in NPM1+/FLT3-ITD(-), NPM1(-)/FLT3-ITD(-), NPM1+/FLT3-ITD+ and NPM1(-)/FLT3-ITD+AML groups (P > 0.05), but there was no significant differences in OS for four groups. CONCLUSIONS: NPM1 and FLT3-ITD mutations are common in AML patients with normal karyotype and related with the clinical characteristics and prognosis of AML.