Evaluation of New Generation Sequencing (NGS)-Based Somatic Gene Variations and Real-Time Polymerase Chain Reaction (PCR)-Based Gene Fusions in Elderly and Young Acute Leukemia Patients: A Retrospective View

İbrahim Halil Erdoğdu 1, Seda Örenay-Boyacıoğlu 2,*, Olcay Boyacıoğlu 3, Nesibe Kahraman-Çetin 1, Füruzan Kacar-Döger 1, İrfan Yavaşoğlu 4, Ali Zahit Bolaman 4

Abstract: Malignant diseases occurring in elderly patients follow a different course from younger patients and show different genetic structures. Therefore, in this retrospective study, the somatic gene variant profile and fusion gene profiles of elderly and young acute leukemia patients were determined to draw attention to the existing genetic difference, and the results were compared. In this study, the records of 204 acute leukemia patients aged 18+ who were referred to the Molecular Pathology Laboratory from the Hematology Clinic between 2018 and 2022 were reviewed retrospectively. Fusion gene detection in patients was performed with the HemaVision®-28Q Panel. The NGS Myeloid Neoplasms Panel was conducted using the MiniSEQ NGS platform according to the manufacturer’s protocol. When all cases are evaluated together, the most frequently diagnosed acute leukemia is acute myeloid leukemia (85.8%). Both groups had a similar fusion gene profile; however, the fusion burden was higher in the elderly group. When the groups were evaluated in terms of somatic gene variations, there were differences between the groups, and the variation load was higher in the elderly group. Considering the different somatic gene variation profiles, it is understood that the genetic structure of tumor cells is different in elderly patients compared to young cases.

Introduction: Malignancies occur at any age, but the incidence of cancer increases with age and is more common in elderly patients. Cancer is the leading cause of death among men and women aged 60–79 years; 60% of newly diagnosed cancers are seen in patients aged 65 and over, and 70% of cancer deaths occur in this age group [1,2]. Due to demographic changes (population aging and increasing life expectancy), the proportion of elderly cancer patients is expected to increase further in the coming decades. It is estimated that 70% of all cancers will afflict patients aged 65 and over by 2030 [2].

According to the World Health Organization (WHO), hematological malignancies constitute 6.5% of all cancers and are responsible for 7.2% of cancer-related deaths [3]. Non-Hodgkin lymphomas (NHLs) are the most common adult hematological malignancies, followed by acute myeloid leukemia (AML), plasma cell diseases, chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL). The median age for NHL, AML, and myeloma is 65, 68, and 69 years, respectively. CML and ALL, on the other side, are generally seen in people in their 50s. In studies evaluating subtypes, it has been shown that the incidence of hematological malignancies increases with age, except for ALL and Hodgkin lymphoma (HL) [4].

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Acute promyelocytic leukemia with PML/RARA (bcr1, bcr2 and bcr3) transcripts in a pediatric patient

Jennifer Santana-Hernández 1,2, Alfredo Corona-Rivera 1,2, Lucero Mendoza-Maldonado 2, Uriel Francisco Santana-Bejarano 2, Idalid Cuero-Quezada 1,2, Aurea Marquez-Mora 2, Graciela Serafín-Saucedo 2, Sinhue Alejandro Brukman-Jiménez 2, Román Corona-Rivera 1,2, Daniel Ortuño-Sahagún 3, Rosa Margarita Cruz-Osorio 4, Fernando Antonio Sánchez-Zubieta 4, Lucina Bobadilla-Morales

Abstract. Patients with acute promyelocytic leukemia (APL) exhibit the t(15;17)(q24.1;q21.2) translocation that produces the promyelocytic leukemia (PML)/retinoic acid receptor α (RARA) fusion gene. Different PML breakpoints yield three alternative molecular transcripts, bcr1, bcr2 and bcr3. The present study reports the simultaneous presence of three PML/RARA transcripts in a pediatric female patient diagnosed with APL, according to the clinical characteristics, immunophenotype and karyotype of the patient. The simultaneous presence of the PML/RARA transcripts were detected using reverse transcription-quantitative PCR (RT-qPCR). This was confirmed with HemaVision-28N Multiplex RT-qPCR, HemaVision-28Q qualitative RT-qPCR and the AmpliSeq RNA Myeloid Panel. To the best of our knowledge, the pediatric patient described in the present study is the first case found to exhibit all three PML/RARA transcripts (bcr1, bcr2 and bcr3). Additionally, a microarray analysis was performed to determine the expression profile, potential predictive biomarkers and the implications of this uncommon finding. According to the information obtained from molecular monitoring, the results reported in the present study were associated with a good patient prognosis. In addition, upregulated genes that are rare in acute myeloid leukemia were identified, and these genes may be promising diagnostic biomarkers for further study. For example, CCL-1 is present in leukemic stem cells, causing treatment failure and relapse, and α- and β-defensins have been reported exclusively in chronic myeloid leukemia. However, the results of the present study confirmed that they may also be present in APL. Thus, these findings suggested a possible signaling pathway that involves the PML/RARA oncoprotein in APL.

Introduction: Acute promyelocytic leukemia (APL) represents ~10% of all childhood acute myeloid leukemia (AML) cases (1,2). Cytomorphologically, APL is classified as hypergranular (or typical M3) or as hypogranular (or variant M3V) (3). APL presents a balanced reciprocal translocation t(15;17)(q24.1;q21.2), involving the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor α (RARA) gene on chromosome 17 (4,5). There are three distinct PML breakpoint cluster regions, namely bcr1, bcr2 and bcr3, that generate a PML/RARA fusion gene of varying mRNA lengths. The bcr1 transcript results from a break in intron 6 of PML with exon 3 of RARA; transcript bcr2 results from a break in exon 6 of PML with exon 3 of RARA; transcript bcr3 results from a break in intron 3 of PML with exon 3 of RARA (6). The frequency of the different breakpoints is dependent principally on ethnicity and geographical location as the bcr1 transcript is more frequent among individuals of Latin origin (3). The aforementioned transcripts have been detected using reverse transcription-quantitative PCR (RT-qPCR) (7,8).

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Google Scholar, 2019

The HemaVision kits are used or mentioned in more than 160 scientific articles.

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Below abstracts from a small fraction of these articles.

Molecular Screening of 28 Genes in Mexican Patients with Acute Myeloid Leukemia a Series of 70 Patients from the Instituto Nacional De Cancerologia, Mexico

Nidia Zapata, Espinoza Ramiro III, Eduardo Cervera, Judith Cruz, Diana Arcos and Juan Labardini

Abstract: Acute Myeloid Leukemia is a clonal heterogeneous disease, where age is an important risk factor to develop theses disease, PCR studies and next generation sequence have proven the diversity of these disease. A lot of genes mutations have been identifying to play a role in the DNA metilation, epigenetics a transcription. We initiate a screening to all acute myeloid leukemias that where the novo or relapse with a 28 gene panel of HEMAVISION a 28q; DNA diagnostic, for the detection al ARN gene fusion and alternatives of the: PML-RAR ALFA (bcr2,V), CBF-MYH11, RUNX1-RUNX1T1, PML-RAR alfa(bcr1,L), KMT2A-MLT3, PML-RAR alfa (bcr3,S), KMT2A-ELL, FUS-ERG, ETV6-MN1, DEK-NUP214, KMT2A-EPS15, KMT2A-AFDN, TCF3-PBX1, ETV6-RUNX1, KMT2A-MLLT1, KMT2A-AFF1, TCF3-HLF, STIL-TAL1, BCR/ABL(p190), SET-NUP214, BCR/ABL(p210), BCR/ABL(p230), ZBTB16-RARalfa, ETV6-ABL1, ETV6-PDGFRB, KMT2A-MLLT10, KMT2A-MLLT11,KMT2A-FOXO4, KMT2A-MLLT6, RUNX1-MECON, NPM1-RAR alfa, NMP1-MLF1, RUNX1-MECON.
FLT3 ITD mutation and D385 by PCR electrophoresis by Invivoscribe was also perform. And the regular cytogenetics and FISH mutations for BCR/ABL, PML/RAR alfa, Inv16, MLL, +8, ETO, BCR, ABL, monosomy 7, monosomy8

Objectives: The main objective is the know the mutation in the Mexican population and the prognostic in these group of patients

Results: These study was perform at Instituto Nacional de Cancerologia, Mexico, randomized patient from 2016-2018 where screen. A total of 70 patients, 37 females and 33 males, ages from 18-85years old, 54 patients where newly diagnosis of acute myeloid leukemia, 4 where relapses and 12 where secondary leukemias, the most frequent FAB morphologic classification where M4:22 cases, M2:15 cases, M3:8 cases, M1:4cases, M0 and M5:3 cases each.
Of the 70 patients: 56 patients where negative to all of the panel screen, FLT3 where only perform in 14 patients 12 where negative and 2 where insufficient to perform the test, the most common FISH translocation was PML/RAR alfa, follow by MLL, ETO and +8.
For the cytogenetics we had 21 cases that didn´t have enough metaphases, 7 normal, 28 cases with more than 2 cytogenetics alterations and 9 with only 1. With a Cytogenetics risk: high risk 44, intermedium:10 and low12.
Of the 70 patient, 14 have some genes mutations +: t(9;11)(p22;q23) KMT2A-MLLT3, t(6;11)(q27;q23) KMT2A-AFDN, t(5;12)(q33;p13) ETV6-PDGFRB, t(8;21)(q22;q22) RUNX1RUNX1T1, inv16(p13q;22q) CBFB-MYH11, t(6;11)(q27;q23) KMT2A-AFDN, t(3;21)(q26:q22) RUNX1-MECOM, inv16(p13q;22q) CBFB-MYH11, t(15;17)(q24;q21) PML-RARA (bcr2,V) t(15;17)(q24;q21) PML-RARA (bcr1,L) t(15;17)(q24;q21) PML-RARA (bcr3,S), t(8;21)(q22;q22) RUNX1RUNX1T1, t(8;21)(q22;q22) RUNX1RUNX1T1, t(15;17)(q24;q21) PML-RARA (bcr3,S)

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Health Technology Assessment of Multiplex Reverse Transcriptase Polymerase Chain Reaction Screening for Translocations at Diagnosis in Acute Myeloid Leukaemia

Haematological Malignancies:
Hans E Johnsen, 1,2 Marianne H Hoffmann, 2 Tobias W Klausen, 2 Ulla H Davidsen, 2 Olav J Bergmann, 2
Martin Boegsted, 1 Mette Nyegaard, 1 Anne Bukh1 and Karen Dybkaer 1
1. Department of Haematology, Aalborg Hospital, Aarhus University Hospital; 2. Herlev Hospital, University of Copenhagen

Abstract: It is predicted that gene diagnostics will become fundamental for future individualised treatment strategies in cancer patients following initial technological investigations and a step-by-step clinical validation. This report is a health technology assessment (HTA) of the use of a novel gene diagnostic assay in acute myeloid leukaemia (AML) with a focus on the technology, the patient, the organisation and economic aspects. In 202 patients with AML, a diagnostic bone marrow or a peripheral blood sample was available for gene analysis, identifying favourable risk translocations by a commercial (HemaVision®) multiplex reverse transcription polymerase chain reaction (RT-PCR) screening assay covering 28 fusion transcripts. The multiplex RT-PCR technology identified five fusion transcripts in five samples not identified by conventional cytogenetics and increased the number of patients identified as having a favourable prognosis from 16 to 21; one translocation identified by cytogenetics was not found by RT-PCR analysis.

Using a prospective questionnaire analysis, a convincing 95% (19/20) patient acceptance was documented for the diagnostic genetic analyses and the outcomes. However, patient understanding requires individual time and dialogue, in accordance with patient rights and modern individualised treatment strategies. The implementation of this strategy may benefit from already existing research laboratories forming a homogenous organisation using standardised technologies, which will make Denmark a high-quality partner in international clinical trials of new therapeutic strategies. The average economic cost per patient analysed using this technique is €600, which represents a modest.

Methods: A total of 329 adult patients (>15 years of age) were diagnosed with AML or chronic myeloid leukaemia (CML) blast crisis at a single institution during a 12-year period between 1 January 1992 and 31 December 2003. Clinical and biological information was collected from patient files for all adult patients with information available (n=321, 98%) to define the study group of 191 samples analysed (see Table 1). In addition, 11 paediatric patients with AML were included prospectively at the time of diagnosis. This patient material has been published previously.1

Conclusion: In conclusion, the technology is effective and complements classic cytogenetic methods. Technologically, evidence-based new gene diagnostic assays in haematology are in a position where they could be introduced tomorrow via already existing laboratories, but not without an increase in expenses for health authorities. However, the increase in cost is limited compared with the cost of new drugs introduced in cancer therapy.

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Combination of broad molecular screening and cytogenetic analysis for genetic risk assignment and diagnosis in patients with acute leukemia.

Leukemia. 2006 Feb;20(2):247-53.
Meyer-Monard S1, Parlier V, Passweg J, Mühlematter D, Hess U, Bargetzi M, Kühne T, Cabrol C, Gratwohl A, Jotterand M, Tichelli A.

Abstract: We evaluated the impact of genetic analysis combining cytogenetics and broad molecular screening on leukemia diagnosis according to World Health Organization (WHO) and on genetic risk assignment. A two-step nested multiplex RT-PCR assay was used that allowed the detection of 29 fusion transcripts. A total of 186 patients (104 males (56%), 174 adults (94%), 12 children (6%), 155 AML (83%), 31 ALL (17%)) characterized by morphology and immunophenotyping were included. Of these 186 patients, 120 (65%) had a genetic abnormality. Molecular typing revealed a fusion transcript in 49 (26%) patients and cytogenetic analysis revealed an abnormal karyotype in 119 (64%). A total of 27 (14%) cases were genetically classified as favorable, 107 (58%) intermediate and 52 (28%) unfavorable. For 38 (20%) patients, there was a discrepancy in the genetic risk assignments obtained from broad molecular screening and cytogenetics. Cryptic fusion transcripts in nine (5%) patients changed the genetic risk assignment in four and the WHO classification in four patients. In 34 patients (18%), cytogenetics defined the risk assignment by revealing structural and numerical chromosomal abnormalities not detected by molecular screening. Broad molecular screening and cytogenetics are complementary in the diagnosis and genetic risk assignment of acute leukemia.

Introduction: Molecular leukemias and chromosomal aberrations are hallmarks of acute leukemia. These have changed our approach to leukemic pathogenesis,1 they have provided a basis for classification, as documented by the World Health Organization (WHO) Classification of Tumors of Haematopoietic and Lymphoid Tissues,2 and they currently guide searches for targeted therapies.3, 4 Therapeutic decision making is based on various well-defined prognostic parameters, the most important being the genetic makeup of the leukemic cell.5, 6 The application of molecular analysis and cytogenetics for diagnosis and

follow-up is controversial. The availabilities of the methods and the need to restrict costs greatly influence the requesting clinician in choosing the approach to genetic characterization. While molecular techniques such as reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH) enable efficient detection of targeted gene abnormalities, cytogenetics provides a comprehensive picture of chromosomal integrity in the leukemic cell without implicating specific genes. It is general practice to search by RT-PCR for AML1-ETO, CBFbeta-MYH11, PML-RARalpha in the case of AML, or for BCR-ABL, E2A-PBX1 and TEL-AML1 in the case of B-ALL. The detection of MLL rearrangements with various partner genes is more laborious. In most cases, a suggestive karyotype is a trigger to perform FISH analysis or Southern blotting. Furthermore, rare fusion transcripts are usually not sought in routine hematology laboratories. Cytogenetics reveals numerical and structural chromosome aberrations, including rare and not yet characterized abnormalities, abnormalities additional to specific rearrangements and complex defects. Although performed routinely, cytogenetic analysis is demanding in terms of time and experience and the quality of the results depends on the expertise of the laboratory.

Results: Distribution of abnormalities and genetic risk assignment
A genetic abnormality was detected in 120 out of 186 patients (65%) using cytogenetics or broad molecular screening or both. In all, 119 patients (64%) had an abnormal karyotype and broad molecular screening revealed a fusion transcript in 49 (26%). Broad molecular screening was positive in 40 (26%) patients with AML and in nine (30%) patients with ALL. Nine cryptic fusion transcripts were detected by broad molecular screening. An abnormal karyotype was found in 96 (62%) AML patients and 23 (74%) ALL patients. A complex karyotype was found in 48 of 186 patients (26%), most of whom (36) had a negative broad molecular screening. In all, 66 (35%) patients had negative results by both methods.

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Broad molecular screening of an unclassifiable myeloproliferative disorder reveals an unexpected ETV6/ABL1 fusion transcript.

Leukemia. 2005 Jun;19(6):1096-9.
Meyer-Monard S, Mühlematter D, Streit A, Chase AJ, Gratwohl A, Cross NC, Jotterand M, Tichelli A.

Abstract: Fusion transcripts are a key element in leukemogenesis. A large number of recurrent chromosomal translocations have been defined, some of which are integrated in the WHO classification of leukemias. Targeted therapies using all-trans-retinoic acid in acute promyelocytic leukemias or the tyrosine kinase inhibitor Imatinib Mesylate in CML have revolutionized the treatment strategies in malignant hematologic diseases.1 Imatinib has been shown to inhibit the tyrosine kinase activity of BCR/ABL, but also of an increasing number of other tyrosine kinases such as ETV6/PDGFRb, c-Kit and FIP1L1-PDGFRa.
The ETV6 gene is located on 12p13 and expressed as a 452 amino-acid protein with two regions of homology to the ETS family of transcription factors. It may be fused to various partner genes mainly coding for tyrosine kinases or transcription factors. In rare cases, it is rearranged with the ABL1 gene, producing two types of oncogenetic proteins with increased tyrosine kinase activity in vitro.2 The low incidence of ETV6/ABL1 gene fusion in leukemia might be attributed to the opposite orientation of both genes necessitating at least three chromosomal breaks to generate a functional fusion gene.3, 4

Introduction: A 65-year-old woman presented in April 2000 with abdominal and thoracic pain. Her history revealed a previous radical hysterectomy for an endometrium carcinoma. No adjuvant chemo- or radiotherapy was given. Laboratory tests showed a white cell count of 22 times 109/l with myeloid precursors, but normal eosinophils, platelets 63

times 109/l and hemoglobin 111 g/l. Bone marrow examination revealed an unclassifiable chronic myeloproliferative disorder. The patient received symptomatic therapy. She was readmitted 1 year later with a pancoast tumor and inguinal lymph node enlargement. Laboratory analysis revealed a leukocytosis of 25.6 times 109/l with 2% blasts and myeloid precursors, hemoglobin 80 g/l and normal platelet count. Bone marrow aspirate and biopsy were hypercellular with abnormal megakaryocytes, an increased number of eosinophils and 10% blasts. Histology of the lymph node was consistent with myeloid sarcoma. The white cell count increased rapidly up to 114.4 times 109/L, with 17% blasts and severe thrombopenia. The patient was treated with low-dose Ara-C, achieved a partial hematological response but developed an ileus and died few days later. No autopsy was performed.


Methods: Cytogenetic analysis of bone marrow cells performed at diagnosis revealed a t(5;9)(q13;q34) as the sole chromosomal abnormality. The constitutional karyotype was normal. RT-PCR for BCR/ABL was negative. Cytogenetic follow-up on bone marrow cells 11 months later revealed the presence of three abnormal clones, all characterized by the presence of t(5;9) and add(17)(q2?1). Additional abnormalities included del(5)(q13;q34) in clone 1, an additional rearrangement involving der(9) in 9q34 in clone 2 and hyperdiploidy (+8, +11, +12, +18 and +19) in clone 3 (Figure 1).

Conclusion: We conclude that broad molecular screening allowed to reveal a genetically distinct entity with poor prognosis, heterogenous morphological and clinical manifestation, but with evident option of a targeted therapy. Broad molecular screening should be an integral part of diagnostics for all types of leukemias. It may detect rare diseases, provide prognostic criteria and clarify therapeutic options early on.

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The added values of multiplex reverse transcriptase-PCR followed by mutation screening in the initial evaluation of acute leukemia.

Int J Lab Hematol. 2016 Aug;38(4):444-53. doi: 10.1111/ijlh.12521. Epub 2016 Jun 20.

Kim B1, Cho YU1, Bae MH1, Jang S1, Seo EJ1, Chi HS1, Park CJ1.
Abstract

INTRODUCTION: This study investigates the benefits of using multiplex reverse transcriptase-PCR (RT-PCR) in addition to standard karyotyping during the initial evaluation of acute leukemia.

METHODS: A total of 1114 consecutive specimens from patients with acute leukemia were tested using a commercial multiplex RT-PCR kit (HemaVision, DNA Diagnostic). NPM1 and CEBPA mutations were selectively tested in acute myeloid leukemia (AML) patients with multiplex RT-PCR negativity.

RESULTS: In specimens with optimal cytogenetics, the frequency of recurrent translocations was 31.3%, and cryptic translocations were detected in 2.1% of samples. The concordance rate between karyotyping and multiplex RT-PCR was 97.5%. In addition to the established functions, we demonstrated the additional benefits of multiplex RT-PCR, including successful molecular characterization, even in cytogenetically suboptimal specimens (5.7%); detection of submicroscopic aberrations (1.0%); detection of rare but potentially significant translocations or variants (2.5%); selection of AML candidates for mutation analysis (68.3%); and finally exclusion of recurrent translocations in patients with acute lymphoblastic leukemia or mixed phenotype acute leukemia (22.5%).

CONCLUSION: We reconfirmed the accuracy and reliability of multiplex RT-PCR for diagnosing acute leukemia and demonstrated additional advantages of this system for the initial evaluation of acute leukemia. Thus, multiplex RT-PCR is worth considering in diagnostic testing of acute leukemias.

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Utility of a multiplex reverse transcriptase-polymerase chain reaction assay (HemaVision) in the evaluation of genetic abnormalities in Korean children with acute leukemia: a single institution study.

Korean J Pediatr. 2013 Jun;56(6):247-53.

Kim HJ, Oh HJ, Lee JW, Jang PS, Chung NG, Kim M, Lim J, Cho B, Kim HK.

Source: Department of Pediatrics, The Catholic University of Korea College of Medicine, Seoul, Korea.
Abstract

PURPOSE: In children with acute leukemia, bone marrow genetic abnormalities (GA) have prognostic significance, and may be the basis for minimal residual disease monitoring. Since April 2007, we have used a multiplex reverse transcriptase-polymerase chain reaction tool (HemaVision) to detect of GA.

METHODS: In this study, we reviewed the results of HemaVision screening in 270 children with acute leukemia, newly diagnosed at The Catholic University of Korea from April 2007 to December 2011, and compared the results with those of fluorescence in situ hybridization (FISH), and G-band karyotyping.

RESULTS: Among the 270 children (153 males, 117 females), 187 acute lymphoblastic leukemia and 74 acute myeloid leukemia patients were identified. Overall, GA was detected in 230 patients (85.2%). HemaVision, FISH, and G-band karyotyping identified GA in 125 (46.3%), 126 (46.7%), and 215 patients (79.6%), respectively. TEL-AML1 (20.9%, 39/187) and AML1-ETO (27%, 20/74) were the most common GA in ALL and AML, respectively. Overall sensitivity of HemaVision was 98.4%, with false-negative results in 2 instances: 1 each for TEL-AML1 and MLL-AF4. An aggregate of diseasesspecific FISH showed 100% sensitivity in detection of GA covered by HemaVision for actual probes utilized. G-band karyotype revealed GA other than those covered by HemaVison screening in 133 patients (49.3%). Except for hyperdiplody and hypodiploidy, recurrent GA as defined by the World Health Organizationthat were not screened by HemaVision, were absent in the karyotype.

CONCLUSION: HemaVision, supported by an aggregate of FISH tests for important translocations, may allow for accurate diagnosis of GA in Korean children with acute leukemia.

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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.

Diagn mol pathol. 2012 Mar;21(1):40-4.

Song MJ, Kim HJ, Park CH, Kim SK, Ki CS, Kim JW, Kim SH

Source: Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Gangnam-gu, Seoul, Korea.
Abstract

Fusion transcripts (FT) from chromosomal rearrangements are key culprits in acute leukemia, with genotype-phenotype correlations including prognostic implications. Here, we report our experience of a commercially available platform utilizing multiplex reverse-transcriptase polymerase chain reaction (RT-PCR), HemaVision, in 309 consecutive patients with acute leukemia. A total of 108 patients (35%) were diagnosed as having acute leukemia with recurrent genetic abnormalities by the World Health Organization 2008 classification. The multiplex RT-PCR platform, detected 12 different FT in 92 (85.2%; 92/108), with a 99% concordance rate with conventional cytogenetics/fluorescence in situ hybridization. Additional information obtained from the multiplex RT-PCR assay included transcript heterogeneity and novel splice variants of FT. In addition, the RT-PCR assay targeting specific FT could be used for monitoring minimal residual disease. HemaVision is a robust diagnostic platform in detecting FT in routine clinical laboratories both at initial diagnosis and for disease monitoring.

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Spectra of chromosomal aberrations in 325 leukemia patients and implications for the development of new molecular detection systems.

J Korean Med Sci. 2011 Jul;26(7):886-92.

Choi HJ, Kim HR, Shin MG, Kook H, Kim HJ, Shin JH, Suh SP, Ryang DW.

Source: Department of Laboratory Medicine, Chonnam National University Hwasun Hospital, Hwasun, Korea.
Abstract

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.

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Diagnostic standardization of leukemia fusion gene detection system using multiplex reverse transcriptase-polymerase chain reaction in Korea.

J Korean Med Sci. 2011 Oct;26(10):1399-400;

Kim MJ, Choi JR, Suh JT, Lee HJ, Lee WI, Park TS.

Comment on Spectra of chromosomal aberrations in 325 leukemia patients and implications for the development of new molecular detection systems. [J Korean Med Sci. 2011]

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Detection of t(3;5) and NPM1/MLF1 rearrangement in an elderly patient with acute myeloid leukemia: clinical and laboratory study with review of the literature.

Cancer Genet Cytogenet. 2010 Jun;199(2):101-9.

Lim G, Choi JR, Kim MJ, Kim SY, Lee HJ, Suh JT, Yoon HJ, Lee J, Lee S, Lee WI, Park TS.

Source: Department of Laboratory Medicine, School of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702, Korea.
Abstract

We present a novel case of acute myeloid leukemia with an NPM1/MLF1 rearrangement in a 78-year-old Korean woman. The bone marrow chromosome study showed a complex karyotype: 46,XX,t(2;13) (q13;q32),der(3)t(3;5)(q25.1;q34),der(5)del(5)(?q31q34)t(3;5),inv(9)(p11q13)c,del(20)(q11.2)[13]/49,idem,+5,+8,+der(13)t(2;13)[7]. Multiplex gene rearrangement testing, cloning, and sequencing analyses revealed an NPM1/MLF1 fusion rearrangement between exon 6 of NPM1 (ENSG00000181163) and exon 2 of MLF1 (ENSG00000178053). Although t(3;5)(q25.1;q34) or the NPM1/MLF1 rearrangement has been reported mostly as a sole karyotypic abnormality in younger patients, it should also be considered in elderly patients with complex chromosomal abnormalities in acute myeloid leukemia or myelodysplastic syndrome.

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Detection of a novel CBFB/MYH11 variant fusion transcript (K-type) showing partial insertion of exon 6 of CBFB gene using two commercially available multiplex RT-PCR kits.

Cancer Genet Cytogenet. 2009 Mar;189(2):87-92.

Park TS, Lee ST, Song J, Lee KA, Lee JH, Kim J, Lee HJ, Han JH, Kim JK, Cho SR, Choi JR.

Source: Department of Laboratory Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea.
Abstract

We report on a 20-year-old man with acute myeloid leukemia (AML) showing a distinct novel CBFB/MYH11 variant fusion transcript. Initial results of bone marrow, chromosome, and flow cytometric analyses were not in accordance with the diagnosis of acute myelomonocytic leukemia with eosinophilia (AML-M4Eo) or AML with a CBFB/MYH11 rearrangement. However, results from 2 commercially available multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) tests repeatedly showed an unusual PCR product from his bone marrow specimen. Not only does this case show a partial insertion of exon 6 of the CBFB (ENSG00000067955) gene, but it also involves novel breakpoints within both exon 6 of the CBFB gene and exon 28 (previously exon 7) of the MYH11 (ENSG00000133392) gene, which is regarded as a previously non-reported, new type (K-type) of CBFB/MYH11 fusion transcript. In addition, our study result was in agreement with the recent report of Schnittger et al. that rare fusion transcripts of CBFB/MYH11 are correlated with an atypical cytomorphology and other aberrant characteristics. Therefore, multiplex RT-PCR and sequence analysis of these atypical products should be performed to diagnose atypical AML with CBFB/MYH11 rearrangement, to predict prognosis of these patients as well as to elucidate the molecular mechanism.

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Comparison of multiplex reverse transcription polymerase chain reaction and conventional cytogenetics as a diagnostic strategy for acute leukemia.

Int J Lab Hematol. 2008 Dec;30(6):513-8.

Source: Department of Hematology-Oncology, Ajou University School of Medicine, Suwon, Korea.
Abstract

To clarify the usefulness of multiplex reverse transcription polymerase chain reaction (mRT-PCR) in diagnosing acute leukemia, mRT-PCR detecting 28 different translocations was performed on bone marrow aspirates of 156 patients with acute leukemia, and the results were compared with conventional chromosomal karyotypes. About 113 of 156 patients had acute myeloid leukemia (AML), and 36 had acute lymphoid leukemia (ALL) with patients' ages ranging from 1 to 84 (median: 34.5). Concordance rate between karyotyping and mRT-PCR was 50% (51% in AML and 44% in ALL). Karyotype revealed chromosomal abnormalities in 70 patients (45%) while mRT-PCR showed some aberrations in 59 patients (38%). mRT-PCR detected t(1;19), t(4;11), t(9;11), t(10;11), t(11;19), t(12;21), and TAL1d, which were not detected by G-banding. In addition, 10 patients with t(15;17), one patient with t(8;21), and four patients with t(9;22) detected by mRT-PCR revealed normal karyotypes. However, mRT-PCR did not detect numerical abnormalities, deletions, and translocations other than the 28 translocations included in the assay as expected. In conclusion, although it cannot be a substitute of the conventional chromosome analysis, mRT-PCR could be a complementary diagnostic strategy of acute leukemia.

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Multiplex reverse transcription polymerase chain reaction screening in acute myeloid leukemia detects cytogenetically unrevealed abnormalities of prognostic significance.

Haematologica. 2005 Jul;90(7):984-6.

Source: Hutchings Hoffmann M, Wirenfeldt Klausen T, Hasle H, Schmiegelow K, Brondum-Nielsen K, Johnsen HE.
Abstract

A commercial multiplex reverse transcription polymerase chain reaction screening assay, covering 28 leukemic fusion transcripts, was applied in 143 samples obtained from patients with acute myeloid leukemia at primary diagnosis. In five patients, a cytogenetically unrevealed fusion gene of prognostic importance was detected, while the assay failed to detect one case of t(15;17).

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Prospective application of a multiplex reverse transcription-polymerase chain reaction assay for the detection of balanced translocations in leukaemia: a single-laboratory study of 390 paediatric and adult patients.

Br J Haematol. 2004 Oct;127(1):59-66.

Olesen LH, Clausen N, Dimitrijevic A, Kerndrup G, Kjeldsen E, Hokland P.

Source: Department of Haematology, Aarhus University Hospital, Aarhus, Denmark.
Abstract

The upfront application of molecular methods for identifying the fusion transcripts arising from balanced translocations in haematopoietic malignancies has several advantages: sensitivity is independent of its frequency, i.e. rare ones are not missed, cytogenetically cryptic aberrations are identified and it provides a platform for minimal residual disease (MRD) detection. Employing a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay identifying 27 fusion transcripts we prospectively analysed blood and/or bone marrow samples from 390 patients referred for diagnosis and treatment for acute leukaemia and chronic myeloproliferative disorders (CMPD) from a geographically well-defined region in Denmark. A total of 233 patients were diagnosed with acute myeloid leukaemia (AML), 95 with acute lymphoblastic leukaemia (ALL) origin and 62 patients were recorded as CMPD. Twenty-three percent AML, 32% ALL and 55% CMPD patients exhibited chromosomal aberrations detected by the multiplex RT-PCR. Cytogenetically cryptic translocations were seen in 15% of the cases. Conversely, the cytogenetic analysis identified chromosomal aberrations other than translocations in 45% of AML cases and 63% of ALL cases. We conclude that, while the fraction of translocation positive leukaemia patients in an unselected cohort is lower than hitherto believed, a molecular approach to their diagnosis is worthwhile, partly for identifying cryptic and rare translocations, partly for monitoring MRD.

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Multiplex RT-PCR for the detection of leukemia-associated translocations: validation and application to routine molecular diagnostic practice.

J Mol Diagn. 2003 Nov;5(4):231-6.

Salto-Tellez M, Shelat SG, Benoit B, Rennert H, Carroll M, Leonard DG, Nowell P, Bagg A.

Source: Department of Pathology, National University of Singapore, Singapore.
Abstract

The aim of this study was to validate the application of a commercially available multiplex reverse transcription polymerase chain reaction (RT-PCR) assay [Hemavision-7 System] for the seven most common leukemia translocations for routine molecular diagnostic hematopathology practice. A total of 98 samples, comprising four groups, were evaluated: Group 1, 16 diagnostic samples molecularly positive by our existing laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 2, 51 diagnostic samples negative by our laboratory-developed assays for PML-RARalpha/t (15;17) or BCR-ABL/t (9;22); Group 3, 21 prospectively analyzed diagnostic cases, without prior molecular studies; and Group 4, 10 minimal residual disease (MRD) samples. Analysis of the two previously studied cohorts (Groups 1 and 2) confirmed the diagnostic sensitivity and specificity of the multiplex assay with regard to these two translocations. Additionally, however, in the "negative" Group (Group 2) the assay revealed three unanticipated translocations (CBFbeta-MYH11, BCR-ABL, and MLL-AF4), two of which were confirmed on cytogenetics. Analysis of the prospective cohort demonstrated that the assay was cost-effective and amenable to standard laboratory practice, with an identically sensitive MRD detection rate to that of our laboratory-developed tests. Virtually all of the results obtained were consistent with the phenotype and karyotype by conventional methods. This study illustrates the utility of a kit-based multiplex RT-PCR assay for the molecular diagnosis and monitoring of leukemias and reinforces the complementary roles of molecular testing and cytogenetics in diagnostic hematopathology.

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Multiplex reverse transcriptase-polymerase chain reaction screening in childhood acute myeloblastic leukemia.

Blood. 2001 Feb 1;97(3):805-8.

Strehl S, König M, Mann G, Haas OA.

Source: Children's Cancer Research Institute (CCRI) and the Ludwig- Boltzmann Institute for Cytogenetic Diagnosis (LBICD), St Anna Children's Hospital, Vienna, Austria.
Abstract

To determine the incidence of leukemia-specific rearrangements, 60 cases of childhood acute myeloblastic leukemia and transient myeloproliferative disorder were screened with a novel multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and the results were correlated with the cytogenetic findings. The RT-PCR assay detects 28 different fusion genes and more than 80 different fusion transcript variants. RNA was isolated from methanol/acetic acid-fixed cells that had been routinely prepared for cytogenetic analysis. Nine different fusion transcripts were found in 40% of the cases, whereas 78.3% of the cases had abnormal karyotypes. Two cases with a t(6;11) and an MLL/AF6 gene fusion were missed cytogenetically. Conversely, cytogenetic analysis revealed 10 other well-defined chromosome rearrangements. Although cytogenetic analysis reveals a much broader range of abnormalities, multiplex RT-PCR serves as quality control and provides the essential information for minimal residual disease studies. Moreover, discrepant findings lead to the detection of new rearrangements on the molecular genetic level.

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