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BRIEF COMMUNICATION
Year : 2017  |  Volume : 13  |  Issue : 6  |  Page : 1042-1046

Detection of balanced translocations in acute lymphoblastic leukemia by a novel multiplex reverse transcriptase reverse transcription-polymerase chain reaction


1 Department of Hematology, Ganga Ram Hospital, New Delhi, India
2 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
3 Department of Clinical Hematology, Army Hospital (Research and Referral), New Delhi, India

Date of Web Publication13-Dec-2017

Correspondence Address:
Dr. Madakshira Gopal Manoj
Department of Pathology, Armed Forces Medical College, Pune - 411 040, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.172128

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 > Abstract 

Fusion transcripts detection is essential for subtyping and diagnosis of acute lymphoblastic leukemia (ALL). This enables institution of appropriate therapy and provides a parameter to monitor disease progression and response to therapy. This study endeared to detect and analyze various balanced translocations known in ALL by using a novel polymerase chain reaction (PCR) method. A pilot study was done in which 16 consecutive cases of ALL were analyzed and followed-up for a period of 1 year. Diagnosis of ALL was established after subjecting blood/bone marrow aspirate samples to morphological examination, immunophenotyping, and detection of fusion transcripts by multiplex reverse transcription (RT)-PCR using HemaVision kit. Results were analyzed by correlating with morphology, immunophenotype, and response to therapy. Epi-Info statistical software was used. 43% (seven cases) showed balanced translocations, with all seven cases being B-ALL and t(9;22) being the most common. There was a consistent association of CD25 cases with t(9;22). Analyses of relation to other parameters were as expected by their respective WHO 2008 subtype. No significant correlation in terms of survival benefit was seen between cases with and without balanced translocations (P = 0.7472). The study demonstrated the utility of multiplex RT-PCR in the initial evaluation, subtyping, and monitoring minimal residual disease in ALL cases with balanced translocations, thereby guiding both therapy and prognosis. The consistent association of CD25 in cases of t(9;22) ALL indicated that CD25 could be used as a surrogate marker.

Keywords: Acute lymphoblastic leukemia, balanced translocations, reverse transcription-polymerase chain reaction


How to cite this article:
Kotwal J, Manoj MG, Kapoor R. Detection of balanced translocations in acute lymphoblastic leukemia by a novel multiplex reverse transcriptase reverse transcription-polymerase chain reaction. J Can Res Ther 2017;13:1042-6

How to cite this URL:
Kotwal J, Manoj MG, Kapoor R. Detection of balanced translocations in acute lymphoblastic leukemia by a novel multiplex reverse transcriptase reverse transcription-polymerase chain reaction. J Can Res Ther [serial online] 2017 [cited 2019 Nov 15];13:1042-6. Available from: http://www.cancerjournal.net/text.asp?2017/13/6/1042/172128


 > Introduction Top


The current pathological approach to the diagnosis of acute lymphoblastic leukemia (ALL) is a multifaceted one involving morphology, cytochemistry, immunophenotyping, cytogenetic, and molecular diagnostic studies.[1] Each component is central to appropriate diagnostic evaluation; however, there is increasing evidence that major disease-defining, prognostically relevant, and therapy-determining data are provided by the latter two genetic analyses. Although conventional cytogenetic studies remain the cornerstone of genetic testing, molecular-based technologies have emerged as a useful tool for the detection of disease-defining genetic lesions.[2] Most translocations, when evaluated at the molecular level, can be detected by reverse transcription-polymerase chain reaction (RT-PCR) in the routine diagnostic setting. RT-PCR detection of the major leukemia translocations has numerous advantages over conventional cytogenetics including shorter turn-around time, no requirement for dividing cells, detection of translocations that may be missed by conventional cytogenetics, and providing a sensitive marker for subsequent minimal residual disease testing.[3] Conventional karyotyping may be hampered by insufficient quality or number of metaphases, low sensitivity, or selective outgrowth. Furthermore, translocations of prognostic significance may be cytogenetically undetected if they involve regions with similar band patterns. The ability to screen for and detect some of the other clinically/therapeutically relevant, leukemia translocations at the molecular level would expand the advantages stated above to a larger number of leukemia specimens.[4] Recognition of patients with abnormalities t(9;22) BCR-ABL chimera gives a bad prognosis to ALL cases. However, recent studies have shown that when patients are given imatinib (tyrosine kinase inhibitor for BCR-ABL) along with chemotherapy, the patients respond very well and go into remission. Thus, molecular testing has important therapeutic implications. The concomitant evaluation of multiple leukemia translocations in the same specimen serves as a powerful internal quality control for assays' analytic specificities and sensitivities. As recognized by the WHO classification, the genetic analysis of leukemia has prognostic and therapeutic implications.[1] This, together with the advent of targeted therapy, underscores the need for an accurate genetic diagnosis that can be rapidly obtained by molecular approaches, and which is key to rational risk stratification and the institution of appropriate therapy. This study endeavors to identify the common balanced translocations associated with ALL and understand their correlation with response to therapy and prognosis.


 > Subjects and Methods Top


A pilot study of 16 consecutive cases of freshly detected ALL from July 2010 to August 2012 was undertaken. The morphological diagnosis of ALL was made according to the FAB classification. Immunophenotyping was done by flow cytometry. Multiplex RT-PCR was performed on all samples to screen for 28 possible translocations using HemaVision kit. The morphology, cytochemistry, immunophenotyping, and RT-PCR results of the cases were collated to classify leukemia as per WHO 2008 guidelines. For morphological examination, phosphate-buffered saline and bone marrow aspirates were air dried and subsequently stained with Romanowsky's stain. Cytochemical stains included myeloperoxidase, which was done in all cases. Bone marrow aspirates and peripheral blood were subjected to immunophenotyping in which the tagged cells were analyzed using BD FACSCalibur system. RNA was extracted from peripheral blood or bone marrow aspirate using Qiagen whole blood RNA extraction kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. Extracted RNA was analyzed using HemaVision multiplex RT-PCR screen test kit (DNA Technology, Aarhus, Denmark) which detects 28 of the most common leukemic fusion genes including 80 splice variants. Reverse transcription was performed with a mixture of translocation-specific primers. PCR amplification was performed in two steps: A master PCR amplification followed by nested PCR, which screened for the presence of fusion transcripts, and a split-out PCR amplification followed by nested PCR which identified the specific fusion transcript. Each of the eight parallel nested multiplex master PCR reactions contained a mixture of primers for the detection of several fusion transcripts, and a primer pair for an internal control gene product of 911 base pair. When the presence of one or more fusion transcripts was detected by the master PCR reactions, the corresponding split-out reactions with individual primers was performed to identify the fusion transcript.


 > Results Top


A total of 16 cases of ALL were diagnosed and evaluated during the period from August 2010 to August 2012, using morphology, cytochemistry, immunophenotyping, and multiplex RT-PCR. All the cases were fresh cases of ALL. Cases of ALL which reported after relapse were excluded. Forty-three percentage (seven cases) of ALLs had balanced translocations, all of which were B-ALL. The most common balanced translocation encountered was t(9;22) [Figure 1]. Correlation with immunophenotyping showed a consistent association of ALL having t(9;22) with CD25. No case of bi-phenotypic leukemia was encountered. The remaining cases showed an immunophenotype as expected of the WHO classification subtype [Table 1]. The mean follow-up period was 11 + 6.132 months (range 2–20). 43.75% (seven cases) succumbed to the illness during the follow-up period. Seventy-five percentage (12 cases) indicated by the absence of blasts in peripheral blood smear. Of the cases who showed a response to treatment responded to treatment, 5 out of 12 cases had a relapse within the follow-up period. Kaplan–Meier survival curve analysis was carried out using log-rank (Mantel–Cox) test. Cases with balanced translocations did not have a significant difference in survival rate, with P= 0.7472 [Figure 2].
Figure 1: Distribution of cases after reverse transcription-polymerase chain reaction analysis

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Table 1: FAB subtype, immunophenotype and PCR result of all cases

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Figure 2: Kaplan–Meir survival graph

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 > Discussion Top


The diagnosis of leukemia starts with morphological examination to document the presence of at least 20% blasts. Cytochemistry which played a central role in older leukemia classification schemes has limited value as per the new WHO guidelines. The basis of initial treatment strategies for ALL depends on immunophenotype, and the presence of any recurrent genetic aberrations. It is thus pertinent that all acute leukemia patients should be screened by cytogenetic or molecular studies. The use of molecular studies, such as multiplex RT-PCR helps to rapidly identify any of the known translocations with the help of multiple primers, without the need for culture or cells in metaphase. At our center, 16 consecutive cases of ALL were evaluated. B-ALL and T-ALL constituted 75% and 25% of cases, which is in agreement with expected incidence.[5] Several congenital risk factors have been identified in the etiopathogenesis of ALL.[6] We had a child who was a known case of Fanconi anemia who developed CALLA-positive B-ALL. 43.75% (seven cases) showed amplification of balanced translocations. The most common translocation amplified was t(9;22) which is in agreement with the previous studies.[4],[7] There were five cases with t(9;22). 80% (four cases) were seen in the pediatric age group, whereas the literature shows a lower incidence in the pediatric population.[8],[9] These cases do not present with lymphadenopathy, as seen in our series.[10] These cases had a B-precursor phenotype of CD10+, CD19+, and TdT+. CD117 was not expressed in all cases. However, CD25 was consistently expressed in all five cases, asserting its association with t(9;22)[11] [Figure 3]. Of the five cases, one patient died. This patient was the only adult among the five cases, highlighting the poor prognosis in adults.[10] Two cases had a relapse; however, all the pediatric cases have responded to imatinib in addition to high-dose chemotherapy and were in remission during the follow-up period.[12],[13] The case with t(11;19) was a 50 years male, who presented with L-2 like blasts with prominent hepatosplenomegaly. Though a high incidence of central nervous system involvement is seen in these cases, which was absent in our case.[10] The blasts had a pro-B phenotype with CD19+, CD20+, and CD10−. MLL gene is known to have many fusion partners of which t(11;19) with ENL gene is seen both in ALL and acute myeloid leukemia cases.[14] Cases with MLL gene rearrangement are known to be associated with a high incidence of FLT-3 mutations.[15] These cases, especially children carry a bad prognosis, and our patient did not go into remission in spite of aggressive management and had a fatal outcome.[16] B-ALL with t(17;19) is a rare alternative translocation involving the E2A (TCF3) gene on chromosome 19.[17],[18] This case was a 5-year-old girl who presented with fatigue and mucosal bleeding. These cases are known to occur in <1% of children with ALL and have a high incidence of developing disseminated intravascular coagulation. The blasts had a pro-B phenotype with CD19+, CD20+, and CD10+. The characteristic association of HLA-DR positivity of the blasts was also seen in our case.[17] Cases with t(17;19) have a dismal prognosis and do not respond to treatment as was in our patient who dead within 3 months of diagnosis.[17]
Figure 3: Illustration of a case of acute lymphoblastic leukemia (a) Peripheral blood picture showing marked increase in blasts (b) High power showing L2 type lymphoblasts with pleomorphic large nuclei

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Multiplex-PCR has various advantages but is also fraught with disadvantages. The entire process involves six steps of amplification and many variables in the form of adequacy of blasts, maintenance of cold chain, the viability of primers, and reagents. However, the presence of the in-built control in each reaction confirmed whether the downstream steps were performed meticulously. The inclusion of cytogenetic analysis would have acted as a good test for correlating our findings and arrive at a more decisive opinion on the utility of the multiplex tandem-PCR assay.

Financial support and sponsorship

Armed Forces Medical Research Project.

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

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Paietta E, Racevskis J, Neuberg D, Rowe JM, Goldstone AH, Wiernik PH. Expression of CD25 (interleukin-2 receptor alpha chain) in adult acute lymphoblastic leukemia predicts for the presence of BCR/ABL fusion transcripts: Results of a preliminary laboratory analysis of ECOG/MRC Intergroup Study E2993. Eastern Cooperative Oncology Group/Medical Research Council. Leukemia 1997;11:1887-90.  Back to cited text no. 11
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