Application of Multiplex RT-PCR for Identification of Leukemia Associated Gene Abnormalities.

Abstract The best prognostic predictor for acute leukemia is known to be the finding of genetic abnormalities of leukemic cells. Methods for detecting the genetic abnormalities include chromosomal studies for karyotyping, FISH(Fluorescence in situ hybridization) and RT-PCR. However, each methods have limitations i.e. low sensitivity in karyotyping, uncertainty of molecular probe to be used in FISH or RT PCR methods. Multiplex RT-PCR (MRT-PCR) allows simultaneous detection of 28 fusion genes, more than 80 breakpoints and splice variants associated with leukemia. Therefore, this method can be used for detection of molecular abnormality in fresh unknown leukemic cases, as well as for molecular remission in follow-up cases. The aim was to demonstrate whether MRT-PCR system might be successfully used to screen a large number of patients with acute leukemia and compare the result with that of chromosome studies. Frozen bone marrow cells from 78 patients, who were diagnosed with acute leukemia at Ajou university hospital between September 1994 and February 2004, were used for MRT-PCR. In all samples with a known conventional cytogenetic results, we performed MRT-PCR and to compared with conventional cytogenetic study regarding the concordance rate and analyzed discordant cases regarding their types. 78 samples(40 male and 38 female patients) were analyzed, and there were 59 AML patients and 19 ALL patients. We successfully obtained the mRNA from all frozen samples. In 21 cases with gene abnormalities by chromosome studies most of them (15/21) showed the same abnormalities with MRT-PCR. In 57 patients with normal karyotype by cytogenetic technique, we identified 18 translocations of clinical significance by MRT-PCR method. In 18 discordant cases, there were 4 cases with t(15;17), 4 cases with t(8;21), 3 cases with t(9;22), 2 cases with t(11;19) and 4 others [t(9;11),t(9;9),t(3;11),inv(16)]. Conventional cytogenetics detected 10 cases of good prognostic gene abnormalities [t(15;17),t(8;21),inv(16)], but MRT-PCR method detected 10 additional cases of good prognostic gene abnormalities which might change the treatment paln. The twenty good prognostic cases with MRT-PCR showed better overall survival than others. (median f/u = 10.6 month, p=0.0443) There were 69% concordance rate between cytogenetic technique and MRT-PCR. Furthermore clinically significant translocations were detected by MRT-PCR in 18 of 57 normal karyotype patients, indicating improved sensitivity and prognostic value with MRT-PCR. Further investigations are needed to ascertain the usefulness of MRT-PCR for the screening tool of leukemic gene abnormalities.

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Diagnostic Utility of a Multiplex RT-PCR Assay in Detecting Fusion Transcripts From Recurrent Genetic Abnormalities of Acute Leukemia by WHO 2008 Classification

Diagnostic Molecular Pathology ◽

10.1097/pdm.0b013e3182319ebe ◽

2012 ◽

Vol 21 (1) ◽

pp. 40-44 ◽

Cited By ~ 7

Author(s):

Min-Jung Song ◽

Hee-Jin Kim ◽

Chang-Hun Park ◽

Sun-Kyung Kim ◽

Chang-Seok Ki ◽

...

Keyword(s):

Acute Leukemia ◽

Diagnostic Utility ◽

Rt Pcr ◽

Pcr Assay ◽

Fusion Transcripts ◽

Genetic Abnormalities ◽

Who 2008

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FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Blood ◽

10.1182/blood.v124.21.5318.5318 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5318-5318

Author(s):

Cristina N. Alonso ◽

Patricia L. Rubio ◽

Adriana Medina ◽

Silvia Eandi Eberle ◽

Andrea Bernasconi ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prognostic Significance ◽

Normal Karyotype ◽

Prognostic Impact ◽

Sequencing Analysis ◽

Rt Pcr ◽

Genetic Abnormalities ◽

Childhood Aml ◽

The Mean ◽

Cebpa Mutations

Abstract Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina. Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting. Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis. Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066). Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. Disclosures No relevant conflicts of interest to declare.

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Distinctive Spectra of Cytogenetic Alterations in Over Five Hundred East Asian Patients with Acute Leukemia

Blood ◽

10.1182/blood.v118.21.4408.4408 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4408-4408

Author(s):

Hyun-Jung Choi ◽

Hye Ran Kim ◽

Myung-Geun Shin ◽

Jong-Hee Shin ◽

Bock-Hee Park ◽

...

Keyword(s):

Acute Leukemia ◽

Chromosomal Aberrations ◽

Molecular Detection ◽

Conflicts Of Interest ◽

Detection System ◽

East Asian ◽

Normal Karyotype ◽

Fusion Genes ◽

Rt Pcr ◽

Conventional Cytogenetics

Abstract Abstract 4408 Background: Racial difference of spectra of chromosomal aberrations in various hematological malignancies was previously reported between Asian and western countries. Therefore, characterization of the cytogenetic profile is still a matter of interests for identifying cytogenetic risk factors in the current risk-adapted chemotherapy. The aim of this study was to investigate the spectrum of chromosomal aberrations in East Asian (Korean) acute leukemia patients, and to propose a panel of leukemic fusion genes suitable for the development of new molecular detection systems. Patients and Methods: We prospectively analyzed bone marrow samples from 502 patients with acute leukemia referred for screening leukemic fusion genes by simultaneous analysis of conventional cytogenetics, FISH and multiplex RT-PCR system in Chonnam National University Hwasun Hospital (Hwasun, Korea) for last 5 years. Results: A total of 334 patients were diagnosed with AML, 155 with ALL and 13 with mixed phenotype acute leukemia. Twenty types in 28 fusion genes were detected in 42.4% of the total patients by multiplex RT-PCR system and in 33.9% by conventional cytogenetics including FISH. In the group of AML, the common fusion genes were 57 PML/RARa, 38 AML1/MGT8, 12 CBFb/MYH11, 11 MLL1, 5 BCR/ABL1, SET/CAN, PLZF/RARA and TLS/ERG in 2 cases each, and one of each AML1/MDS1, TEL/ABL and TEL/MN1. In the group of ALL, the following fusion genes were detected: 33 BCR/ABL1, 22 TEL/AML1, 10 MLL1, 7 E2A/PBX, 2 SET/CAN and SIL/TAL, one TEL/ABL. In addition, cytogenetically cryptic translocations were detected in 14.8% patients with normal karyotype or numerical aberrations by conventional cytogenetics. Among 288 patients with negative results by multiplex RT-PCR system, one child with early pre-B-ALL harbored MLL1/AF4, and 8 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10), which were all confirmed by conventional cytogenetics. Conclusions: The current study demonstrated the spectrum and frequency of chromosomal aberrations in Korean patients with acute leukemia, which are different from Caucasian data. Also these results may offer important implications for the development of new multiplex molecular detection system. Disclosures: No relevant conflicts of interest to declare.

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Analysis of Genetic Alterations In 843 Korean Acute Leukemia Patients by Multiplex RT-PCR Assay (HemaVision) and Karyotyping

Blood ◽

10.1182/blood.v116.21.4845.4845 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4845-4845

Author(s):

Jihyang Lim ◽

Myungshin Kim ◽

Yonggoo Kim ◽

Kyungja Han ◽

Hee-Je Kim ◽

...

Keyword(s):

Acute Leukemia ◽

Conflicts Of Interest ◽

Simultaneous Detection ◽

Normal Karyotype ◽

Genetic Alterations ◽

Rt Pcr ◽

Pcr Assay ◽

Abnormal Karyotype ◽

Acute Leukemias ◽

Positive Results

Abstract Abstract 4845 Background: The importance of molecular tests has been increased for diagnosis, classification and MRD monitoring in acute leukemia. Recently, multiplex RT-PCR assays (HemaVision) for simultaneous detection of 28 leukemia-associated translocations was introduced and has been used for molecular screening of leukemic patients. We analyzed the results of multiplex RT-PCR assay (HemaVision) and karyotyping in Korean acute leukemia patients. Methods: From April 2007 to March 2010, multiplex RT-PCR assay (HemaVision, DNA Technology A/S, Denmark) was performed in 843 Korean acute leukemia cases (AML 566 vs. ALL 277 and adults 596 vs. pediatrics 237) for screening leukemia-associated translocations. All results were compared with chromosome analysis results by conventional karyotyping. Results: Three hundreds fifty-one cases (41.6%) were positive in multiplex RT-PCR. In AML cases, 221 cases (39.0%) revealed positive results and RUNX1-RUNX1T1 85 cases (38.5%), PML-RARA 74 cases (33.5%), CBFB-MYH11 25 cases (11.3%) and MLL-related 19 cases (8.6%) were frequently detected. In contrast, 130 cases (46.9%) revealed positive results in 277 ALL cases and BCR-ABL1 68 cases (52.3%), ETV6-RUNX1 28 cases (21.5%), TCF3-PBX1 15 cases (11.5%), MLL-related 10 cases (7.7%) were frequently detected. In comparison to karyotyping, 337 cases (96.0%) revealed abnormal karyotype and 14 cases (4.0%) revealed normal karyotype in 351 cases that revealed positive results by multiplex RT-PCR assay. In 492 cases that revealed negative results by multiplex RT-PCR assay, 278 cases (56.5%) revealed abnormal karyotype and 214 cases (43.5%) revealed normal karyotype. Overall 629 cases (74.6%) were detected genetic alterations by multiplex RT-PCR assay and/or karyotyping. Conclusion: Most acute leukemias may have a genetic alteration in their leukemogenesis. Both multiplex RT-PCR assay and karyotyping are essential for screening leukemia-associated genetic alterations. Disclosures: No relevant conflicts of interest to declare.

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Diagnosis of helicobacter pylori infection: problems and prospects

Medical alphabet ◽

10.33667/2078-5631-2020-17-54-59 ◽

2020 ◽

pp. 54-59

Author(s):

A. S. Molostova ◽

N. S. Gladyshev ◽

A. V. Svarval ◽

R. S. Ferman ◽

A. B. Karasyova ◽

...

Keyword(s):

Helicobacter Pylori ◽

Urease Activity ◽

Diagnostic Methods ◽

Study Group ◽

Biochemical Method ◽

Antimicrobial Drugs ◽

Non Invasive ◽

Number Of Patients ◽

Pcr Method ◽

Positive Results

(HP) infection was performed using invasive and non-invasive methods. The study group consisted of 95 patients with dyspepsia. HP infection was detected in 47 patients (49.4 %). The expediency of using a set of diagnostic methods for detecting HP (PCR, immunochromatographic, bacteriological and method for determining urease activity) is proved. Most often (100 %) in patients HP infection was detected in biopsies using the PCR method. Somewhat less frequently it was detected when examining biopsies with an invasive biochemical method (AMA RUT Reader) (82 %) and fecal immunochromatographic method (83 %). Despite the fact that helicobacteriosis was detected bacteriologically in a small number of patients (24 %), this method is of particular value, since it allows you to assess the sensitivity to antimicrobial drugs and probiotics, and does not give false positive results.

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Real-time Quantitative RT-PCR method for estimation of mRNA level of CCAAT/enhancer binding protein in the central nervous system ofLymnaea stagnalis

Acta Biologica Hungarica ◽

10.1556/abiol.55.2004.1-4.19 ◽

2004 ◽

Vol 55 (1-4) ◽

pp. 157-161 ◽

Cited By ~ 7

Author(s):

D. Hatakeyama ◽

Hisayo Sadamoto ◽

E. Ito

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Real Time ◽

Binding Protein ◽

Mrna Level ◽

Rt Pcr ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein ◽

The Central Nervous System ◽

Pcr Method

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Investigation of Antibacterial Effect and Expression of Alkaline Protease Gene in Pseudomonas Aeruginosa (PAO1) Using Copper Oxide, Copper Complex and Satureja Khuzistanica: RT-PCR Method

International Journal of Pharmaceutical Research ◽

10.31838/ijpr/2020.12.04.067 ◽

2020 ◽

Vol 12 (04) ◽

Keyword(s):

Pseudomonas Aeruginosa ◽

Copper Oxide ◽

Copper Complex ◽

Alkaline Protease ◽

Antibacterial Effect ◽

Protease Gene ◽

Rt Pcr ◽

Pseudomonas Aeruginosa Pao1 ◽

Pcr Method ◽

Alkaline Protease Gene

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SARS-CoV-2 in peritoneal swabs from asymptomatic patients undergoing emergency abdominal surgery

Journal of Surgical Case Reports ◽

10.1093/jscr/rjab116 ◽

2021 ◽

Vol 2021 (4) ◽

Author(s):

Jasim AlAradi ◽

Rawan A Rahman AlHarmi ◽

Mariam AlKooheji ◽

Sayed Ali Almahari ◽

Mohamed Abdulla Isa ◽

...

Keyword(s):

Peritoneal Fluid ◽

Case Series ◽

Rapid Test ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Protective Measures ◽

The Public ◽

Asymptomatic Patients ◽

Number Of Patients ◽

Viral Transfer

Abstract This is a case series of five patients with acute abdomen requiring surgery who tested positive for coronavirus disease 2019 (COVID-19) and were asymptomatic, with the purpose of detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in peritoneal fluid. Nasopharyngeal swab was done as a prerequisite for admission or prior to admission as part of random testing. Two methods of viral testing were employed: Xpert® Xpress SARS-CoV-2 (rapid test) and real-time reverse transcription polymerase chain reaction (RT-PCR). Either or both tests were done, with the former performed for patients requiring surgery immediately. Surgery was performed within 24–36 h from admission. Peritoneal fluid swabs were obtained for the detection of SARS-CoV-2 using RT-PCR test. Swabs were immediately placed in viral transfer media and delivered to the public health laboratory in an ice bag. SARS-CoV-2 was not detected in peritoneal swabs. Due to the limited number of patients, further studies are required; yet, protective measures should still be taken by surgeons when dealing with COVID-19 cases.

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