Detection of NPM1 Mutation in Acute Myeloid Leukemia by Droplet Digital PCR and Its Clinical Application Value / 中国实验血液学杂志

OBJECTIVE@#To establish the droplet digital PCR (ddPCR) assay for the detection of NPM1 type A mutation in patients with acute myeloid leukemia (AML), and to evaluate its specificity, sensitivity and its value in clinical application.@*METHODS@#NPM1 mutant and wildtype plasmids were used to verify the performance of ddPCR. Both ddPCR and Sanger sequencing were used to detect the bone marrow samples of 87 AML patients, which were confirmed by next generation sequencing (NGS). Moreover, NPM1 mutation burden was dynamically monitored in five patients by ddPCR.@*RESULTS@#The limit of blank (LOB) of ddPCR established for NPM1 mutation detection was 1.1 copies/μl, and the limit of detection (LOD) was 2.43 copies/μl, which had good linearity. Among the 87 newly diagnosed AML patients, ddPCR identified seventeen cases positive for NPM1 mutation (19.5%)， which was consistent with Sanger sequencing. NGS confirmed 12 positive cases, including 8 of type A mutations, 2 of type D mutations, and 2 of rare type mutations. The results of dynamic monitoring of NPM1 mutation burden in 5 patients showed that the NPM1 mutation burden decreased obviously even close to 0, when patients achieve complete remission after chemotherapy. However, the mutation burden was increased again at the time of relapse.@*CONCLUSION@#In this study, we established a ddPCR method for detection of NPM1 mutation with good sensitivity and repeatability, which can be used for screening NPM1 mutation in newly diagnosed AML patients and for minimal residual disease monitoring after remission in positive AML patients to guide treatment.

Human papillomavirus genotypes in male patients attending the STD clinic in Zhenjiang area / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the status of human papillomavirus ( HPV) infection and its genotypes in male patients in Zhenjiang area.</p><p><b>METHODS</b>Using PCR and reverse dot blot hybridization, we determined the genotypes of HPV DNA in 245 male patients at our Clinic of Dermatology and STD.</p><p><b>RESULTS</b>The total rate of HPV infection was 43.67% (107/245), and 18 subtypes were detected. Among the 107 HPV-positive cases, low-risk, high-risk, and combined high- and low-risk infections accounted for 39.25% (42/107), 38.32% (41/107), and 22.43% (24/107), respectively. The most notable low-risk HPV types were HPV6 and HPV11, and the most notable high-risk HPV types were HPV16, HPV52, and HPV58. The rates of single infection and multi-infection were 53.27% (57/107) and 46.73% (50/107), respectively. One case had the most types, infected with 8 genotypes. No statistically significant differences were observed in the total rate of HPV infection among different age groups (Χ2 = 7.999, P > 0.05).</p><p><b>CONCLUSION</b>The dominant subtypes of HPV infection in male patients in Zhenjiang area were HPV6, HPV11, and HPV16. The most common subtypes were HPV6 and HPV11 in low-risk infection, and HPV16, HPV52, and HPV58 in high-risk infection.</p>

Gene expression of helicase antigen in patients with acute and chronic myeloid leukemia / 中国实验血液学杂志

The aim of this study was to investigate the expression status of the helicase antigen (HAGE) transcript and its clinical significance in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). The expression of HAGE cDNA in bone marrow mononuclear cells from AML and CML patients was detected by using real-time quantitative PCR. The results indicated that overexpression of HAGE transcript (117.12% - 9842.70%, median 434.96%) was detected in 14.8% (11/74) AML patients. AML patients with HAGE cDNA expression were significantly older than those HAGE-negative patients (median 67 and 45 years, respectively, p = 0.001). HAGE cDNA expression was more frequently present among the patients with acute monoblastic leukemia (M(4) and M(5), 7 of 20, 35.0%), compared to the patients with acute non-monoblastic leukemia (M(1), M(2), M(3) and M(6), 4 of 54, 7.4%) (p = 0.007). 28.6% (8/28) cases with normal karyotypes showed HAGE cDNA overexpression, significantly higher than 7.5% (3 of 40) in those with chromosomal abnormalities (p = 0.041). Overexpression of HAGE transcript was found in 9 (34.6%) CML cases and more frequently observed at accelerated phase and blast crisis (4/4, 100%) than that at chronic phase (5/22, 22.7%) (p = 0.008). It is concluded that HAGE cDNA expression is relevant to specific subtypes of AML and to the progression of CML.

Scanning of c-kit gene mutations in acute myeloid leukemias using high-resolution melting analysis / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect the common mutations (D816V and N822K) of c-kit gene in acute myeloid leukemia (AML) using high-resolution melting analysis (HRM).</p><p><b>METHODS</b>HRM analysis was established to screen c-kit mutations in PCR products of c-kit exon 17 in 21 AML patients with t(8;21). PCR products were sequenced to confirm the mutation.</p><p><b>RESULTS</b>HRM analysis identified an aberrant melting curve in 6 cases (28.6%), which were confirmed by direct DNA sequencing as one D816V mutation and five N822K mutation.</p><p><b>CONCLUSION</b>HRM analysis is a convenient, rapid, specific and high-throughput technique for scanning c-kit gene mutation in AML.</p>

Alteration of methylation status of death-associated protein kinase (dapk) gene promoter in patients with acute myeloid leukemia / 中国实验血液学杂志

This study was purposed to analyze the methylation status of death-associated protein kinase (dapk) gene promoter in Chinese patients with acute myeloid leukemia (AML) and its relationship with clinical features. The methylation-specific PCR (MSP) technique was used to detect dapk promoter methylation in bone marrow samples from 112 cases of AML. The results indicated that gene dapk promoter hypermethylation was detected in 82 cases (73.2%), but not in 13 control group. There was no correlation of dapk gene hypermethylation with sex, age, WBC counts, platelet counts, hematologic parameters, chromosomal abnormalities and different subtypes of AML patients. It is concluded that dapk gene hypermethylation may be a common molecular event in AML.

Quantification of GRAF gene expression in patients with acute myeloid leukemia using EvaGreen real time quantitative PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To quantify the expression level of GRAF gene in acute myeloid leukemia (AML) and analyze its clinical significance.</p><p><b>METHODS</b>The EvaGreen real-time quantitative polymerase chain reaction (RQ-PCR) assay was established and performed to measure the GRAF gene transcripts in 71 cases with AML and 21 with nonmalignant hematological diseases. The clinical correlation of GRAF expression was analyzed.</p><p><b>RESULTS</b>The established EvaGreen RQ-PCR assay had good specificity, reproducibility and sensitivity. The GRAF expression level was significantly lower in AML (0.01%-169.75%, median 3.82%) than that in controls (14.49%-126.85%, median 56.04%) (P<0.05). There was no correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups (P>0.05).</p><p><b>CONCLUSION</b>The GRAF gene was down-regulated in AML, which might play a role in the leukemogenesis.</p>

Quantification of prame gene transcript in chronic myeloid leukemia / 中国实验血液学杂志

This study was purposed to analyze the expression level of preferentially expressed antigen of melanoma (prame) transcript in the patients with chronic myeloid leukemia (CML) and explore its clinical significance. Real-time quantitative PCR (RQ-PCR) assay was used to detect the level of prame gene transcript in the bone marrow samples from 30 patients with CML and 15 patients with iron deficiency anemia (IDA). The results showed that CML patients had significantly higher prame mRNA level (0% - 772.25%, median 8.28%) than IDA cases (0% - 1.46%, median 0.19%) (p < 0.001). The level of prame gene transcript was significantly correlated with that of bcr-abl fusion gene transcript (r = 0.708, p < 0.001) in CML patients. Furthermore, 6 patients in blastic crisis (BC) and accelerated phase (AP) had significantly higher prame gene transcript than that of 24 cases in chronic phase (CP) (p = 0.007). In 2 CML patients with sequential samples, prame gene transcript increased in AP and BC, compared with in CP, and was consistent with the altering tendency of bcr-abl transcript. It is concluded that the level of prame gene transcript increases in CML which associates with the progression of the disease, prame gene transcript level can be used for monitoring the disease state.