Dynamic monitoring of plasma circulating DNA in patients with acute myeloid leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to quantify plasma circulating DNA level in patients with acute myeloid leukemia (AML) and to evaluate its clinical significance. 66 AML patients and 100 controls (60 healthy subjects for health examination, 20 cases of benign hematopathy, and 20 cases of solid tumors) were enrolled in this study. Blood samples were collected from AML patients at different status of disease and control groups. Circulating DNA were drew by using the BILATEST DNA Kit. The level of plasma DNA was determined by using duplex real-time quantitative PCR. The results showed that the median value of plasma DNA level in AML patients at diagnosis was 168.5 (73.4 - 245.1) ng/ml, significantly higher than those in three control groups, and the median level in male patients was significantly higher than that in female patients (P = 0.019). No significant difference was found in plasma DNA level of the patients at different ages and with different FAB subtypes. Compared with level before chemotherapy, the plasma DNA levels in complete remission patients and partial remission patients decreased significantly, and with no statistical difference from level of healthy controls, but was significantly different from level of non-remission patients (P < 0.05). Following up of 31 remission patients showed that the plasma DNA level increased in 5 out of 6 (83.3%) relapsed patients, but no increase was found in 22 out of 25 (88.0%) non-relapsed patients. It is concluded that the quantification of plasma DNA may be useful for evaluating therapeutic effects and monitoring relapse in AML patients.

Efficacy of chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab for chronic lymphocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of combination chemoimmunotherapy of fludarabine, cyclophosphamide and rituximab (FCR) in chronic lymphocytic leukemia (CLL).</p><p><b>METHODS</b>Twenty-one patients with CLL were treated with FCR regimen which consisted of fludarabine (25 mg/m(2), days 2 to 4), cyclophosphamide (250 mg/m(2), days 2 to 4) and rituximab (375 mg/m(2), day 1) in a course of 28 days. The minimal residual disease (MRD) was determined by multiparameter flow cytometry. The correlation between the pretreatment characteristics and complete remission (CR) rate was analyzed.</p><p><b>RESULTS</b>Eleven patients (52.4%) achieved CR, 7 (33.3%) achieved partial remission (PR) with a overall response (OR) rate of 85.7%. With a median follow-up time of 19 (7 - 73) months, the overall survival (OS) was 86.0%, and the progression-free survival (PFS) was 72.0%. Pretreatment parameters independently associated with higher CR rates were Binet stage A + B, IgVH mutated and ZAP-70 less than 20%. MRD was less than 1% in 6 patients. The most common toxicities were myelosuppression and gastrointestinal reaction.</p><p><b>CONCLUSION</b>FCR is an effective regimen for CLL patients.</p>

Analysis of DNMT3a gene mutations in acute myelogenous leukemia / 中国实验血液学杂志

This study was purposed to investigate the mutational status of DNA methyltransferase (DNMT3a) gene and the clinical features of AML patients with DNMT3a mutations. Using PCR combined with directly sequencing, the somatic mutations of DNMT3a involving residue of amino acid 882 were detected in 77 AML patients. Furthermore, the clinical features of these patients were also studied. The results showed that the DNMT3a mutation were detected in 7 out of 59 patients with de novo AML (11.9%), which included 4 patients with DNMT3a R882C, 2 patients with DNMT3a R882H and 1 patient with DNMT3a Y874C. Morphology examination indicated that 2 patients were M(2), 1 patient was M(4) and 4 patients were M(5). Cytogenetic analysis revealed that karyotype in 5 out of 7 patients with DNMT3a mutation were normal. In total of 27 patients with normal karyotype 5 patients (22.7%) were found harboring DNMT3a mutation, while no DNMT3a mutation was found in 21 patients with abnormal karyotype. The mutation rate in patients with positive CEBPA was obviously higher than that in patients with negative CEBPA (p = 0.002). Immunophenotype analysis showed that 4 patients (4/7, 57.1%) with DNMT3a mutation expressed lymphoid antigens including CD4 or/and CD7. There were no statistical significance in age, gender, blast cells of bone marrow, white blood cell and platelet counts, hemoglobin level, ratio of CR, mutations of FLT3-ITD, NPM1 and c-kit between patients with DNMT3a mutation and patients with wild DNMT3a (p > 0.05). It is concluded that the DNMT3a mutations are more prevalent in AML patients with normal karyotype accompanying with positive NPM1 and/or CEBPA mutation, the role of DNMT3a mutation in AML prognosis needs to be further studied.

Comparison of conventional cytogenetics and interphase fluorescence in situ hybridization in diagnosis of acute promyelocytic leukemia / 中国实验血液学杂志

The aim of this study was to explore cytogenetic characteristics of acute promyelocytic leukemia (APL) and compare the interphase fluorescence in situ hybridization (I-FISH) with conventional cytogenetic (CC) analysis. A total number of 157 APL patients were recruited in this study, and the I-FISH and CC were applied to analyze cytogenetic features. Chromosome samples of bone marrow cells were prepared by short-term culture. Out of all 157 cases, 136 were observed with CC assay, 66 with I-FISH, of which 45 samples were analyzed with both methods. The results showed that among all 136 CC samples, t(15;17)(q22;q21) was found in 120 cases, of which 107 cases was isolated t(15;17)(q22;q21) abnormality, 13 cases was complex abnormalities and 12 case without mitotic figure. Among all 66 cases of I-FISH group, PMI/RARα fusion gene was found in 64 cases (97.0%), suggesting that I-FISH group was more sensitive than CC group (p = 0.041). It is concluded that combination of I-FISH and CC techniques plays a pivotal role for diagnosis and detection of minimal residual disease in APL.

mRNA levels detected by real-time quantitative RT-PCR in chronic lymphocytic leukemia and their clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression level of murine double minute 4 (MDM4) mRNA in chronic lymphocytic leukemia (CLL) and its prognostic value in CLL. By means of β-actin as internal reference, the real-time quantitative RT-PCR was set up. The expression of MDM4 mRNA in 66 CLL patients was measured by fluorescence dye SYBR Green I. The dispersion of MDM4 expression ratio of groups with different prognostic factors was described by using Mann-Whitney U test. The results showed that the median MDM4 mRNA expression level was 0.037098 (0.088245-0.014875) in CLL patients. The expression level of MDM4 mRNA was significantly higher in patients with P53 gene deletion than that in patients without P53 gene deletion (0.13167 vs 0.030927) (p < 0.001), and also significantly higher in patients with P53 mutation than that in patients without P53 mutation (0.13167 vs 0.03077) (p < 0.001). MDM4 expression was also associated with Binet stages (p = 0.044) and ATM gene deletion (p = 0.046), but was not associated with LDH (p = 0.216), β(2)-MG (p = 0.314), TK1 (p = 0.300), ZAP-70 (p = 0.559), CD38 (p = 0.513) and IgVH mutation status (p = 0.333). It is concluded that the expression level of MDM4 is significantly higher in patients with P53 deletion or mutation. MDM4 expression is significantly associated with Binet stages and ATM gene deletion. MDM4 may be an important prognostic factor in CLL.

Prognostic significance of telomere length in patients with chronic lymphocytic leukemia / 中国实验血液学杂志

This study was aimed to explore the prognostic significance of telomere length in patients with chronic lymphocytic leukemia (CLL) and to analyze relation of telomere length with Binet stage, IgVH mutation status, CD38, ZAP-70 expression as well as other clinical features. 35 CLL patients who contained 80% or more tumor cells in the peripheral blood or bone marrow samples were selected as objects studied, while 13 healthy donors were served as normal controls. The telomere relative length was detected by using a real-time fluorescent quantitative polymerase chain reaction method (qPCR); the expression of CD38 and ZAP-70 protein were detected by flow cytometry, the IgVH mutation was detected by multiplex PCR. The results showed that the mean telomere relative length in CLL patients and normal controls were 0.384 and 0.443 respectively, but the difference between them was not significant (p > 0.05). The telomere length was significantly correlated with Binet stages and IgVH mutation status. Patients in Binet stage B and C showed significantly shorter telomeres than those in Binet stage A (p = 0.001). Mean telomere relative lengths in patients without IgVH mutation were shorter than those in patients with IgVH mutation (p = 0.015). No relation of telomere length with sex, age, ZAP-70 protein and CD38 were found (p > 0.05). It is concluded that telomere length may have a prognostic significance for CLL patients. Combining telomere length and IgVH mutation status may achieve a better prognostic subclassification for CLL patients.

Detection of miRNA levels in leukemia patients by real-time quantitative PCR / 中国实验血液学杂志

This study was purposed to establish a method for detecting miRNA expression by using fluorescent quantitative polymerase chain reaction (RQ-PCR), and to investigate the application value of this method in quantitative detection of miRNA. Total RNA was extracted from the peripheral blood or bone marrow of 82 patients with chronic lymphocytic leukemia (CLL) and 70 patients with acute leukemias (AL). Standard curves of U6 and miRNA were constructed from 10-fold serial dilutions of the cDNA, the quantitative detection was performed by using real-time quantitative PCR with SYBR Green by Roche Light Cycler. U6 RNA was used as the reference, the relative expression levels of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in patients with CLL, while miR-128-1, miR-223, let-7b, miR-155 and miR-181a in patients with AL were analyzed by 2((-DeltaDeltaCT)) method. The relative parameters including specificity, linearity range, sensitivity and repeatability were evaluated. The results showed that the RQ-PCR assay could precisely detect the mature miRNA in as few as 10-50 ng of total RNA with the sensitivity of 0.05 ng RNA. Ct values of miRNA and U6 were all within 15 to 30. A good linear relationship (R(2) > 0.980) was obtained from the standard curves, also the efficiency of amplification was above 90%. Only a specific peak was shown on the melting curve diagram. The coefficients of variation (CV) of interrun and intrarun assay was < 4.8% and 6.3% respectively. It is concluded that the RQ-PCR is a sensitive and fast method for the detection of miRNA with its high-flux and low cost, this method will facilitate the research with detection of miRNA.

Detection of puma mRNA levels by real-time quantitative RT-PCR in chronic lymphocytic leukemia and its clinical significance / 中国实验血液学杂志

This study was aimed to investigate the expression level of puma (p53 up-regulated modulator of apoptosis) mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. The puma mRNA expressions in 100 CLL patients and 11 normal controls were measured by relative quantification RT-PCR with fluorescent dye SYBR Green I, the beta-actin was used as internal reference. The difference of puma expression rate between groups with different prognostic factors was described using the Mann-Whitney U test. The relative quantitative value of puma expression was calculated by means of 2 (-ΔCt). The results indicated that the correlation coefficients of the standard curves in qRT-PCR were ≥ 0.99. The coefficients of variations (CV) within group or between groups were < 5%, and the sensitivity reached 10² copies/microg RNA. The median puma mRNA expression level was 1.038 x 10⁻³ (4.106 x 10⁻⁴ - 2.806 x 10⁻³) in CLL patients, which was 1.220 x 10⁻³ (7.233 x 10⁻⁴ - 1.405 x 10⁻³) in normal controls. There was no difference of puma mRNA expression between CLL patients and normal controls (U = 544.5, p = 0.957). Puma expression was significantly correlated with Binet stages (p < 0.001), expression of CD38 (p = 0.002), ZAP-70 protein (p = 0.012), LDH levels (p = 0.009) and beta₂-MG (p = 0.046). The puma expression level in patients with earlier Binet stage (Binet stage A) was obviously higher than that in patients with later Binet stage (Binet stage B, C). The puma expression levels in patients with positive expression of CD38 and ZAP-70 protein, elevating levels of LDH and beta₂-MG were sharply lower than those in patients without above-mentioned unfavorable factors. The puma expression was also correlated with molecular cytogenetic abnormalities, the puma expression levels in patients with trisomy 12 (p = 0.003) and 14q32 translocation (p = 0.045) detected by FISH were significantly lower than those in patients without above-mentioned molecular cytogenetic abnormalities. It is concluded that the qRT-PCR assay is reliable and sensitive. Puma mRNA expression is significantly correlated with a great deal of prognostic factors, and may be a prognostic marker of CLL.

Clinical and biological features of 8 patients with Richter's syndrome / 中国实验血液学杂志

In order to evaluate the clinical, biological features and prognostic factors of Richter's syndrome (RS), 8 RS patients were analyzed retrospectively. The serological test, multiplex parameter flow cytometry, conventional cytogenetic analysis, FISH technique and PCR combined with sequence detection were used to detect the LDH, β(2)-MG, TK1, SF, CA125, ZAP-70, chromosome karyotype, ATM and p53 gene deletion, as well as +12 abnormality and IgVH mutation. The results indicated that 7 out of 8 patients transformed to diffuse large B cell lymphoma (DLBCL) and 1 patient transformed to Hodgkin lymphoma (HL). Among 8 patients, LDH level in 7 patients, β(2)-MG level in 4 patients, SF level in 7 patients, CA-125 level in 4 patients and TK1 level in 1 patient exceeded the normal range. Meanwhile, ZAP-70 and CD38 were expressed positively in 4 and 7 out of 8 patients respectively. Unmutated IgVH was found in 5 patients, and 4 patients had the complex chromosome abnormalities. +12 and p53 deletion was found in 1 patient. 8 patients were divided into two groups (Binet A + B and Binet C), the mean time from diagnosis to progression was 98.5 months in Binet A + B group, compared with 38.3 months in Binet C group, there was significant difference between two groups (p = 0.021). Mean overall survival was 123.8 months and 49.8 months in two groups, respectively (p = 0.049). The mean survival after transformation was 34.5 months in Binet A + B group and 10.3 months in Binet C group. In conclusion, the level of LDH, β(2)-MG and SF are higher in RS patients in Binet C group, and so are the incidence of high expressed ZAP-70 and CD 38, unmutated IgVH. The clinical stage may be the risk and prognostic factors for RS transformation.

Correlation of deoxycytidine kinase gene expression with fludarabine resistance in patients with chronic lymphocytic leukemia / 中国实验血液学杂志

The study was purposed to explore the relationship between the expression of deoxycytidine kinase (dCK) gene and fludarabine (Flud) resistance in patients with chronic lymphocytic leukemia (CLL). The real time quantitative polymerase chain reaction (RQ-PCR) technique was used to detect the mRNA expression of dCK gene in the bone marrow or peripheral blood mononuclear cells from 30 CLL patients, the difference of dCK expression levels between CLL patients sensitive to Flud and patients resistant to Flud was analyzed. The results showed that dCK expression level in the Flud-sensitive patients was much higher than that in the Flud-resistant ones (p = 0.001); dCK expression level had no relationship with sex, age, Binet stage, IgVH mutations, CD38, ZAP-70 or p53 gene mutation (p > 0.05). It is concluded that low expression or deficiency of dCK in patients may contribute to resistance against Flud- and the prognostic significance of dCK expression remains to be further studied.

High-dose methylprednisolone for the treatment of refractory chronic lymphocytic leukemia / 中国实验血液学杂志

To investigate the curative effects of high-dose methylprednisolone (HDMP) in the treatment of refractory chronic lymphocytic leukemia (CLL), 5 patients with CLL who poorly reacted to several cycles of fludarabine based protocols with or without rituximab were treated with 1 to 6 cycles of HDMP with 1 g/(m(2)xd) for d1-5. All the patients were at Binet stage C. 3 patients were at Rai stage IV and 2 were at Rai stage III. 2 patients were diagnosed as Richter syndrome. CD38 and ZAP-70 were expressed in 5 and 3 patients respectively. All the patients developed with B group symptoms including fever, night sweat and/or weight loss and so on. Clinical manifestations and complete blood cell count, peripheral blood smear, bone marrow aspirate, hepatic and renal function, blood serum electrolytes, blood glucose were examined, CD5(+)CD19(+) lymphocytes of peripheral blood and bone marrow were determined by flow cytometry. The results showed that B group symptoms disappeared in 4 patients at 2 - 4 weeks after therapy. The size of enlarged lymph nodes was reduced in all the 5 patients. In 1 patient spleen was not palpable from 10 cm below costal margin at 2 weeks after therapy, and his hemafecia was alleviated. The renal function in another patient with renal failure recovered to normal after two cycles of therapy. Pancytopenia improved in 3 patients after therapy. CD5(+)CD19(+) lymphocytes decreased in all the patients. 4 patients acquired partial remission and 1 patient acquired stable status of disease. The side effects became mild. In conclusion, the therapeutic results preliminarily show that HDMP is an effective and safe protocol for the treatment of refractory CLL.

Abnormality of serum immunoglobulin in peripheral blood of patients with chronic lymphocytic leukemia / 中国实验血液学杂志

In order to investigate the relation of serum immunoglobulin (Ig) level to age, sex, disease stages and prognosis in chronic lymphocytic leukemia (CLL) patients, the levels of serum IgG, IgA and IgM in 83 CLL patients were detected by immune rate turbidimetry. the expressions of CD38 and ZAP-70 in CLL cells were determined by multi-parameter flow cytometry (FCM). The results showed that among the 83 CLL patients, the IgG, IgA and IgM levels were reduced in 12 (14.5%), 26 (31.3%) and 34 cases (41.0%) respectively. The incidence of Ig reduction was higher in Binet C stage than that in Binet A and B (p=0.011). There was higher incidence of Ig reduction in high-risk group of Rai stage than that in low-risk group (p=0.011). The positive rate of CD38 or ZAP-70 was significantly higher in Ig reduction group than that in normal Ig level group (p=0.033 and p=0.038 respectively). The positive rate of CD38 and ZAP-70 was also higer in advanced disease, and among them the positive rate of ZAP-70 expression in Binet C stage was obviously higer than Binet A and B (p=0.047). Nonetheless, there was no significant relationship of Ig level with gender and age (p>0.05). It is concluded that the function of humoral immunity in CLL patient is closely related to the disease stages. The serum Ig detection seems important for evaluating immunologic state and prognosis of CLL patients.

Analysis of chromosome karyotype abnormality in acute myeloid leukemia / 中国实验血液学杂志

This study was aimed to investigate the characteristics of chromosome karyotype abnormality in patients with acute myeloid leukemia. 379 cases of de novo acute myeloid leukemia were enrolled in this study. Chromosome preparations were made on bone marrow cells by using direct method or short-term culture. Chromosome karyotypes were analyzed by R-banding technique. The results indicated that 216 out of 379 patients had clonal chromosome aberrations with the percentage of 56.99%, including 19 kinds of balanced translocations and 70 kinds of chromosome gain or loss. The most common structure and numerical abnormalities were t(15;17) and -Y with the percentage of 25.86% and 5.80%, respectively. -Y was accompanied by t(8;21) in 90.9% of the -Y abnormality cases, which accounted for 40.81% of t(8;21) positive cases. The abnormality of M(3) was significantly higher than the other FAB subtypes (p < 0.05). No significance was found between the male and female groups for the chromosome aberrations (p > 0.05). In conclusion, some specific chromosome aberrations are correlated with specific FAB subtype, which may contribute to the clinical diagnosis and subtyping of the disease.

Cytogenetic analysis of 362 cases of chronic myeloid leukemia / 中国实验血液学杂志

In order to evaluate the cytogenetic features and clinical significance of chronic myeloid leukemia (CML), chromosome preparation of bone marrow cells was made by using 24-hour culture, and R-banding technique was employed for karyotyping in 362 patients with CML. The patients were divided into two groups of chronic phase (CP) and blast crisis (BC). The results showed that the incidence of additional chromosome, variant translocation and Philadelphia (Ph) negative, bcr/abl positive CML with abnormal chromosomes in CP group were 70 cases (26.1%), 19 cases (7.1%), 4 cases (1.5%), and were 50 cases (53.2%), 8 cases (8.5%), 4 cases (4.3%) in BC group. Among the 362 cases, 324 cases (89.5%) were Ph positive. Classic translocation was found in 297 cases (91.7%) and variant translocation in 27 cases (8.3%), including 13 cases of simple variant, 13 cases of complex variant and 1 case of marked Ph. Special karyotypes were found in 120 out of 362 cases. Analysis of these karyotypes demonstrated that the most common numerical abnormalities were +Ph (21.7%), +8 (10.0%), +21 (10.0%), +19 (7.5%) and structure abnormalities were i(17q) (13.3%). In conclusion, compared to chronic phase, the incidence of additional chromosome, variant translocation and so on are much higher at in blast crisis. It is feasible to evaluate the progress of the disease by karyotype analysis.

Deletions of derivative chromosome 9 in 138 patients with chronic myeloid leukemia / 中国实验血液学杂志

To investigate the frequency of derivative chromosome 9 [der (9)] deletions in patients with chronic myelogenous leukemia (CML), karyotype analysis in 138 patients with CML was performed with R-banding technique, and dual fusion fluorescence in situ hybridization (FISH) was used to detect der (9) deletion. The results showed that among 138 cases, 126 cases were Ph positive (91.3%) from which 122 cases were typical Ph translocation, 12 cases were Ph negative (8.7%). FISH detection revealed 23 with der (9) deletions out of 138 cases (16.7%), 20 out of 122 cases with typical Ph translocation showed typical Ph translocation (16.4%) and 3 out of 4 cases with variant Ph translocation had variant Ph translocation (75%). 20 cases were in chronic phase (CP) (17.2%), 3 cases were in blast crisis (BC) (17.6%), there was no significant difference in the frequency of the der (9) deletions between the cases in CP and in BC (p < 0.05). It is concluded that incidence of der (9) partial deletions in CML patients is 16.7%, FISH can effectively detect the der (9) deletions, and there is no correlation of der (9) deletion frequency between cases in different phases of CML.

Detection of the Philadelphia chromosome in adult B-lineage acute lymphoblastic leukemia using dual-color dual-fusion interphase fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the incidence of Philadelphia chromosome (Ph) in adult B-lineage acute lymphoblastic leukemia (B-ALL).</p><p><b>METHODS</b>One hundred and twelve adult patients with previously untreated B-ALL were prospectively investigated by interphase dual-color dual-fusion fluorescence in situ hybridization (DD-FISH) with two-color break apart probe BCR-ABL and the results were compared with that of conventional cytogenetics (CC).</p><p><b>RESULTS</b>The incidence of Ph chromosome was 17.98% (16/89) and 31.25% (35/112) by CC and DD-FISH, respectively. The mean positive rate of Ph+cells by FISH was 66.23% (ranging 18.5%-99%). Of the 35 Ph+ALL patients by FISH, 25 were successfully karyotyped by CC which included 5 normal karyotypes, 20 abnormal karyotypes including 16 Ph chromosome and 13 complex abnormalities.</p><p><b>CONCLUSION</b>The incidence of Ph chromosome was 31.25% in adult with B-ALL. DD-FISH with BCR-ABL probe provides a powerful technique for the diagnosis of Ph+B-ALL. It is an important supplement to the CC analysis. DD-FISH technique should be used as a routine method for the diagnosis for adult acute B-ALL.</p>

Immunoglobulin variable heavy chain region genetic constitution and mutation status in Chinese patients with chronic lymphocytic leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the genetic constitution and mutation status of the immunoglobulin variable heavy chain region (IGVH) gene expression in Chinese patients with chronic lymphocytic leukemia (CLL).</p><p><b>METHODS</b>The IGVH mutation was detected by multiplex PCR and direct sequencing of the purified PCR products from 64 CLL patients. The segments of VH, DH and JH family and mutations were analyzed by IMGT/V-QUEST and IGBlast.</p><p><b>RESULTS</b>In the 64 patients, the most common usage was VH3 (31/64, 48%), followed by VH4 (26/64, 41%), VH1 (4/64, 6%), VH2 (2/64, 3%) and VH7 (1/64, 2%). The results also showed that 44 patients (69%) had mutated VH, 6 cases (9%) had identical germline sequences. Among the 64 sequences of DH segments, DH3 gene family was used most frequently (25/64, 39%), among which 11 cases had unmutated VH. The most frequent usage of the JH segments was JH6.</p><p><b>CONCLUSION</b>There is significant difference in the frequency of the IGVH gene family in Chinese CLL patients compared to Western patients, suggesting the involvement of antigen selection in different ethnic and/or environmental factors in CLL disease initiation, and its prognostic significance needs further investigation.</p>

The application of fluorescence in situ hybridization in detecting chronic myeloid leukemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the clinical significance of the application of fluorescence in situ hybridization (FISH) in detecting chronic myeloid leukemia (CML).</p><p><b>METHODS</b>Chromosome preparation was made by using 24-hour culture. FISH technique using dual color dual fusion (DC-DF) BCR/ABL probe was performed in all 158 cases and R-banding was also employed for karyotyping in some patients.</p><p><b>RESULTS</b>Among the 158 cases, 98 cases were Ph positive, of which 69 cases (70.4%) were typical FISH pattern (1R1G2F), the other 29 cases (29.6%) showed 12 different types of atypical FISH pattern. The most frequent atypical patterns found were 1R1G1F in 7 cases (7.1%), 2R1G1F in 5 cases (5.1%), 1R1G2F and 1R1G3F in 4 cases (4.1%), 2R2G1F in 3 cases (3.1%). Karyotype analysis on 18 CML cases with atypical FISH patterns demonstrated that the atypical FISH patterns were due to variant translocation in 3 cases; the additional third signal was because of a supernumerary Ph chromosome. The karyotyping results did not conform to FISH results in four cases suggesting the conceivable mistakes in karyotyping. The 1R1G1F signal pattern seen in 3 cases with classical t(9;22) resulted from the deletion of derivative chromosome 9. The 1R1G2F signal pattern detected in 40% to 64% of interphase cells of 3 cases without Ph chromosome by conventional cytogenetic analysis suggested a submicroscopic translocation. Three cases treated with Glivec or bone marrow transplantation showed normal karyotypes with a small amount of BCR/ABL positive cells by FISH detection.</p><p><b>CONCLUSION</b>FISH technique is of great value for the diagnosis of CML and confirmation of variant translocation, occult Ph translocation, derivative chromosome 9 deletion, therapeutic effect of interferon and Glivec as well as detection of minimal residual disease after bone marrow transplantation.</p>

Lipoprotein lipase and serum thymidine kinase level in chronic lymphocytic leukemia and their correlations with other prognostic factors / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate lipoprotein lipase (LPL) and serum thymidine kinase (TK) levels in chronic lymphocytic leukemia (CLL) and their correlations with other prognostic factors.</p><p><b>METHODS</b>LPL expression level in peripheral blood samples of 58 CLL patients was detected by semi-quantitative reverse transcription PCR (RT-PCR). Serum TK1 level in 39 CLL patients was detected by enhanced chemiluminescence (ECL) and TK monoclonal antibody (Anti-TK mAb). IgVH mutation status was detected by multiplex PCR and sequencing of purified PCR products. The expression of ZAP-70 protein and CD38 were determined by flow cytometry . Panel probes and fluorescence in situ hybridization (FISH) were used to detect cytogenetic aberrations.</p><p><b>RESULTS</b>The median LPL expression level in CLL was 0.26 (0 -6.29), while undetectable in normal controls. LPL expression level was significantly correlated with IgVH mutation status, Binet stages, CD38 and cytogenetic aberrations. Patients with unmutated IgVH genes had higher LPL than those with IgVH mutations (P = 0.010). Patients in Binet stage B and C had higher LPL than those in stage A (P = 0.011). LPL level was higher in patients with CD38 > or = 30% (P = 0.001). Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) than those with favorable cytogenetics (deletion in 13q as the sole abnormality) (P = 0.002). LPL level was not significantly correlated with sex, age, and ZAP-70 protein (P > 0.05). The level of TK1 was higher in CLL patients than that in normal control (P < 0.05). Patients with higher level of absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), unmutated IgVH genes and ZAP-70 had higher levels of TK1 than those with lower level of ALC, LDH, mutated IgVH genes and ZAP-70 (P = 0.018, P = 0.018, P = 0.030 and P = 0.038, respectively). TK1 level had no correlation with sex, age, Binet stages, CD38, and cytogenetic aberrations (P > 0.05).</p><p><b>CONCLUSIONS</b>LPL expression and serum TK1 levels significantly correlate with other CLL prognostic factors and could be predictive markers for IgVH mutation status. LPL and serum TK1 might be applied to the assessment of prognosis in CLL patients.</p>

Prognostic factors for chronic lymphocytic leukemia with typical and atypical immunophenotype / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the prognostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, absolute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations.</p><p><b>METHODS</b>According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophenotype CLL, 16 with score 4 or 3 were atypical immunophenotype CLL. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, sIg, CD20, CD79b expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations.</p><p><b>RESULTS</b>There was no significant difference between the two groups in sex, age, ZAP-70 and IgVH mutation status (P=0.398, P=0.189, P=0.268 and P=0.131, respectively). The incidence of ALC> or =50 x 10(9)/L, Binet B + C, CD38> or =30% in atypical CLL patients (43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P=0.026, P<0.01 and P=0.026, respectively). The proportion of typical patients (26.8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P=0.022).</p><p><b>CONCLUSION</b>There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.</p>