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

2012 ◽  
Vol 21 (1) ◽  
pp. 40-44 ◽  
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

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4431-4431
Author(s):  
Hyun Woo Lee ◽  
Youn Mu Jung ◽  
Jun Ho Jang ◽  
Jun Seong Park ◽  
Sung Ran Cho ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Normal Karyotype ◽  
Concordance Rate ◽  
Molecular Probe ◽  
Rt Pcr ◽  
Prognostic Gene ◽  
Number Of Patients ◽  
Genetic Abnormalities ◽  
Pcr Method ◽  
Cytogenetic Technique

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.

scholarly journals Multiplex RT-PCR Assay for Detection of Common Fusion Transcripts in Acute Lymphoblastic Leukemia and Chronic Myeloid Leukemia Cases

2016 ◽  
Vol 17 (2) ◽  
pp. 677-684 ◽  
Author(s):  
Nittaya Limsuwanachot ◽  
Teerapong Siriboonpiputtana ◽  
Kanlaya Karntisawiwat ◽  
Takol Chareonsirisuthigul ◽  
Suporn Chuncharunee ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts

scholarly journals INCIDENCE OF COMMON FUSION TRANSCRIPTS IN ADULT AND PEDIATRIC ACUTE MYELOID LEUKEMIA (AML) CASES: EXPERIENCE OF A TERTIARY CARE RESEARCH INSTITUTE

2012 ◽  
Vol 4 (1) ◽  
pp. e2012042 ◽  
Author(s):  
PRATEEK Bhatia ◽  
Jogeshwar Binota ◽  
Neelam Varma ◽  
RK Marwaha ◽  
Pankaj Malhotra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Tertiary Care ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Prognostic Information ◽  
Pediatric Acute Myeloid Leukemia ◽  
Fusion Transcripts ◽  
The Common ◽  
Pediatric Aml

Introduction: The incidence of common fusion transcripts in AML is 40-45%, but data from Indian sub-continent is limited. Aims & Objectives: The aim of the present study is to note the incidence of common fusion transcripts of AML1-ETO, PML-RARA and CBFβ-MYH11 in adult and pediatric AML cases. Materials & Methods: A total of 116 AML cases diagnosed on bone marrow, cytochemistry and Flow-cytometry over a period of 1.5 year were enrolled and bone marrow samples in EDTA were processed by Multiplex RT-PCR assay. Results: Of 116 cases, 96 (83%) were adult and 20 (17%) pediatric cases. A total of 39/116 (33.6%) cases showed positivity for fusion transcripts of which 28/96 (29.16%) were adult and 11/20 (55%) pediatric cases. Of the 28 positive adult cases, 14/96 (14.58%) were positive for AML1-ETO, 12/96 (12.5%) for PML-RARA and 2/96 (2.08%) for CBFβ-MYH11. In the 11 positive pediatric cases, 6/20 (30%) were positive for AML1-ETO, 3/20 (15%) for PML-RARA and 2/20 (10%) for CBFβ-MYH11. Discussion & Conclusion: The incidence of the common fusion transcripts in our pilot study is in accordance with that described in western studies. It is important to identify these transcripts as they provide useful prognostic information to the treating clinician.

scholarly journals A STUDY ON THE EXPRESSION OF BCR-ABL TRANSCRIPT IN MIXED PHENOTYPE ACUTE LEUKEMIA (MPAL) CASES USING THE REVERSE TRANSCRIPTASE POLYMERASE REACTION ASSAY (RT-PCR) AND ITS CORRELATION WITH HEMATOLOGICAL REMISSION STATUS POST INITIAL INDUCTION THERAPY

2012 ◽  
Vol 4 (1) ◽  
pp. e2012024 ◽  
Author(s):  
Prateek Bhatia ◽  
Jogeshwar Binota ◽  
Neelam Varma ◽  
Deepak Bansal ◽  
Amita Trehan ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Treatment Protocol ◽  
Rt Pcr ◽  
Pediatric Case ◽  
Post Induction ◽  
The Status ◽  
Low Platelet Count ◽  
Mixed Phenotype ◽  
Hematological Remission ◽  
Who 2008

Introduction: The MPAL comprise 2-5% of all acute leukemia. The present WHO 2008 classification has separated two groups in MPAL based on t(9;22) positivity and MLL rearrangement. Aims & Objectives: The aim of the present pilot study is to note the incidence of BCR-ABL transcript in MPAL cases using the RT-PCR assay and to correlate the status with hematological remission post induction. Materials & Methods: A total of 10 MPAL cases classified on Flow-cytometry based on the current WHO 2008 criteria were enrolled. In all the cases Bone marrow or peripheral blood sample in EDTA was processed for molecular studies and the RT-PCR reaction carried out using primers specific to the t (9;22) and t(4;11) translocation. The post induction check marrow slides were also reviewed. Results: Out of the total 10 MPAL cases, 7/10 (70%) were adult and 3/10 (30%) pediatric cases. A total of 4/10 (40%) cases showed positivity for the t(9;22) transcript and none for t (4;11). Of the 4 positive cases, 3/10(30%) were adult cases and 1/10(10%) pediatric case. The BCR-ABL transcript type in adult cases was b3a2 (p210) in 2/3 (66%) and e1a2 (p190) in 1/3 (33.3%) case. The single pediatric case was positive for b3a2 transcript. Discussion & Conclusion: All the 4 positive MPAL cases presented with high TLC and low platelet count (p<0.05). The positive cases also showed hematological remission at post induction check marrow (blasts<5%). This could partly be explained due to good response to the imatinib added to the treatment protocol.

scholarly journals Quantitative analysis of TEL/AML1 fusion transcripts by real-time RT-PCR assay in childhood acute lymphoblastic leukemia

Leukemia ◽  
2000 ◽  
Vol 14 (8) ◽  
pp. 1526-1528 ◽  
Author(s):  
P Ballerini ◽  
J Landman Parker ◽  
I Laurendeau ◽  
M Olivi ◽  
M Vidaud ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Acute Lymphoblastic Leukemia ◽  
Real Time ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts

The Molecular Screening Using Multiplex Reverse Transcription Polymerase Chain Reaction and the Comparison with Cytogenetic Analysis in the Prognostic Stratification of Acute Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4868-4868
Author(s):  
Young-Uk Cho ◽  
Hyun-Sook Chi ◽  
Seongsoo Jang ◽  
Eul-Ju Seo ◽  
Jung-Hee Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Acute Leukemia ◽  
Cytogenetic Analysis ◽  
Risk Group ◽  
Molecular Screening ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Fusion Transcripts ◽  
Prognostic Stratification ◽  
Polymerase Chain

Abstract INTRODUCTION: The detection of chromosomal aberrations is essential for the diagnosis and prognostic stratification of acute leukemia. Classical cytogenetic analysis has the advantage of comprehensive screening of all types of abnormalities such as numerical aberrations and deletions as well as translocations. However, this technique is time-consuming and labor-intensive, and cannot detect cryptic translocations. Recently, a multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) has emerged as a tool for overcoming disadvantages of cytogenetic analysis with some degree of comprehensiveness. We designed multiplex RT-PCR system for acute leukemia based on sets of chromosomal translocations rationally selected with cost-effectiveness and clinical relevance. Herein, we tried to use molecular screening of acute leukemia with a multiplex RT-PCR system we designed, and compared with the results of cytogenetic analysis in terms of prognostic stratification. MATERIALS AND METHODS: A total of 121 consecutive patients with acute leukemia were included from November 2006 to July 2007. Multiplex RT-PCR system (Seegene, Korea) for molecular screening was carried out to detect 16 fusion transcripts: BCR-ABL, PML-RARα, PLZF-RARα, AML1-ETO, CBFβ-MYH11, DEK-CAN, E2APBX1, TEL-AML1, and MLL rearrangements (MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ELL, MLL-ENL, MLL-AF17, MLL-PTD). Cytogenetic analysis was performed by standard GTL-banding technique. The discordant findings were validated by fluorescence in situ hybridization (FISH) or conventional single RT-PCR. RESULTS: Eighty-nine (73.6%) adults and 32 (26.4%) children were included in this study. A total of 77 (63.6%) were diagnosed as acute myeloid leukemia (AML) and the remaining 44 (36.4%) were acute lymphoblastic leukemia (ALL) including 7 biphenotypic acute leukemia. Cytogenetic analysis was available in 118 (97.5%) of the patients. Sixty-four cases of fusion transcripts were detected in 60 patients (49.6%). Within the AML group, the following fusion transcripts were detected: 14 AML1-ETO, 8 PML-RARα, 3 CBFβ-MYH11, 1 DEK-CAN, 1 BCR-ABL, 6 MLL-PTD, 4 MLL-AF6, 3 MLL-AF9, 1 MLL-ELL. Within the ALL group, the following fusion transcripts were detected: 12 BCR-ABL, 4 TEL-AML1, 2 E2A-PBX1, 3 MLL-AF4, 1 MLL-AF9, 1 MLLPTD. The concordance rate between two tests was 88.3%. In 14 cases, the results of two methods did not agree. Seven cytogenetically unrevealed abnormalities were detected with the multiplex RT-PCR: 3 TEL-AML1, 2 MLL-AF9, 1 MLL-AF6, 1 PML-RARα. The frequency of cryptic translocation was 5.8%. MLL-PTD which is impossible to be observed by cytogenetic analysis was detected in remaining seven patients. On the other hand, the corresponding fusion transcripts were not detected by multiplex RT-PCR in three cases of inv(16), t(4;11), and MLL rearrangement suggested by FISH, respectively. Molecular screening could preferentially assign 32.5% of AML patients to favorable-risk group, and did 34.1% of ALL patients to adverse-risk group. Although the concordance rate between two methods as a prognostic factor was 89.5% in risk-determined group by multiplex RT-PCR, six patients (3 AML and 3 ALL) disclosed different risk group by molecular screening from cytogenetic analysis. CONCLUSIONS: We conclude that molecular screening using multiplex RT-PCR was useful in providing informations about genetic abnormalities more rapidly and giving appropriate therapeutic decision in acute leukemia patients. It was also useful in the detection of cytogenetically cryptic translocations. Although the multiplex RT-PCR system could not be a whole substitute for cytogenetic analysis, we expect that two methods are complementary at the time of diagnosis of acute leukemia, by which more genetic aberrations would be detected efficiently.

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

Blood ◽  
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.

Update on the large-scale screening of ALK fusion oncogene transcripts in archival NSCLC tumor specimens using multiplexed RT-PCR assays.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 7594-7594 ◽  
Author(s):  
Tianhong Li ◽  
Eric Huang ◽  
Sonal Desai ◽  
Laurel Beckett ◽  
Craig Stephens ◽  
...  
Keyword(s):  
Large Scale ◽  
Fusion Gene ◽  
Standard Of Care ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Gene Transcripts ◽  
Pcr Assays ◽  
Alk Positive ◽  
Large Scale Screening

7594 Background: The ALK inhibitor crizotinib offers a new standard of care for advanced NSCLC patients with EML4-ALK fusion oncogenes. We previously reported a 4.0% frequency of EML4-ALK fusion oncogene transcripts detected in 1889 NSCLC specimens in the RGI database (Li et al., ASCO 2011). Methods: Patented single and multiplexed RT-PCR assays suitable for rapid and accurate detection of all variants of ALK fusion oncogene transcripts were used as previously described, including all 9 known EML4-ALK fusion gene transcripts and ALK RNA levels (Danenberg, ASCO 2010). The sensitivity and specificity on archival formalin-fixed, paraffin-embedded tumor specimens are 99% and 100%, respectively. We here update the detection of EML4-ALK fusion transcripts in the RGI database. Results: Between 12/2009 and 09/2011, 4750 NSCLC specimens in the RGI database were tested for the presence of ALK fusion transcripts. We found 152 (3.2%) NSCLC cases with EML4-ALK fusion positivity, including 87 (57.2%) V1, 15 (9.9%) V2, 47 (30.9%) V3, and 3 (2.0%) V5a variants. Median age (range): 61.1 (33-96). Female: 74 (49%). All EML4-ALK-positive tumors were adenocarcinomas. No EGFR or K-Ras mutation was detected in ALK fusion-positive samples. Expression of chemotherapy-related biomarkers was available from 63 (female: 31, 49%) EML4-ALK-positive cases in the database: 43 (68%) had low TS level of <2.33; 40 (63.5%) had low ERCC1 level of <1.7, and 25 (40%) had low RRM1 level of <0.97. Conclusions: This RT-PCR assay provides a tool for rapid, large-scale screening of NSCLC FFPE tissues for EML4-ALK fusion gene transcripts. The relative value of this RT-PCR assay as a companion diagnostic test for drugs targeting ALK merits evaluation in comparison with the FDA approved ALK FISH test.

scholarly journals Screening and Quantification of Multiple Chromosome Translocations in Human Leukemia

2003 ◽  
Vol 49 (7) ◽  
pp. 1066-1073 ◽  
Author(s):  
Run Zhang Shi ◽  
Joseph M Morrissey ◽  
Janet D Rowley
Keyword(s):  
Fusion Gene ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fusion Transcripts ◽  
Chromosome Translocations ◽  
Pcr Multiplex ◽  
Pcr Products ◽  
Gene Transcripts ◽  
Genetic Analyzer

Abstract Background: Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays. Methods: We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts. Results: The multiplex RT-PCR assay enabled screening of &gt;10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100–10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay. Conclusions: The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.

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