Mediterranean Journal of Hematology and Infectious Diseases Incidence of Common Fusion Transcripts in Leukemia (aml) Cases: Experience of a Tertiary Care Research Institute

Introduction: The incidence of common fusion transcripts in AML is 40 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 Materials & Methods: A total of 116 AML cases diagnosed on bone marrow, cytochemistry and Flow-cytometry over a period of 2 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 p (12.5%) for PML-RARA and 2/96 (2.08%) for CBF 6/20 (30%) were positive for AML1 MYH11. Discussion & Conclusion: The incidence of t accordance with that described in western studies. It is important to identify these transcripts as they provide useful prognostic information to the treating clinician.

The incidence of common fusion transcripts in AML is 40-45%, but data from Indian The aim of the present study is to note the incidence of common fusion ETO, PML-RARA and CBFβ-MYH11 in adult and pediatric AML cases. A total of 116 AML cases diagnosed on bone marrow, cytochemistry and cytometry over a period of 2 year were enrolled and bone marrow samples in EDTA were PCR assay. 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 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 CBF 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.

45%, but data from Indian
The aim of the present study is to note the incidence of common fusion t and pediatric AML cases. A total of 116 AML cases diagnosed on bone marrow, cytochemistry and cytometry over a period of 2 year were enrolled and bone marrow samples in EDTA were 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%) ositive for AML1-ETO, 12/96 MYH11. In the 11 positive pediatric cases, RARA and 2/20 (10%) for CBFβhe common fusion transcripts in our pilot study is in accordance with that described in western studies. It is important to identify these transcripts as group as the universal criteria for 20% blasts is not necessary for diagnosis of leukemia, if any of the group are present. Moreover, the different genetic alterations imply prognostic information to the treating clinician regarding the disease behavior and outcome. Though most of the recurrent translocations can be detected using conventional karyotyping or Fluorescent in hybridization (FISH) technique, but these are either cumbersome or costly and depend upon good quality metaphases. Multiplex Reverse transcriptase PCR is a relatively cost effective and sensitive procedure to detect the common fusion transcripts and can be utilized as an initial diagnostic screening assay in AML cases.
Though many western studies have highlighted the incidence of common fusion transcripts in AML, the data from our sub-continent is limited. The aim of the present pilot study is to note the incidence of the common chimeric fusion transcripts of AML1-ETO, PML-RARA and CBFβ-MYH11 in adult and pediatric AML cases using the multiplex RT-PCR assay.
Methodology. The present study is a prospective study carried out over a period of 2 years (April 2010-March 2012) in the Department of Hematology of a tertiary care and referral institute (PGIMER, Chandigarh) of Northern India. The study involved 116 cases of AML diagnosed on bone marrow examination, cytochemistry and immunophenotyping. In each case a 2-3 ml Bone marrow sample was collected for multiplex RT-PCR assay. Total RNA was extracted from the sample using the commercial kit (Qiagen Miniamp RNA blood Kit-50 Reactions) according to the manufacturer's instructions and then reverse transcriptase reaction was performed using the cDNA synthesis kit (Fermantas). The quality of cDNA was analyzed using the primers for beta-actin housekeeping gene. The multiplex RT-PCR assay was then carried out using primers specific (see Table 1 for primer sequences & product size) for each of the above transcripts as per the protocol designed by S.Pakakasama et al 1 in their study. In addition to the above Multiplex RT-PCR, another Multiplex RT-PCR was carried out separately in suspected AML-M3 cases to detect all the three isoforms of PML-RARA i.e. Bcr 1 (intron 6): Long isoform; Bcr 2 (exon 6): Variable isoform and Bcr 3 (intron 3): Short isoform (see Table 1 for primer sequences and PCR product sizes for the primer pairs). This PCR assay was carried out in following conditions-Pre-Denaturation-95c-1 mt (one cycle); Denaturation-94c-1mt; Annealing-65c-1 mt; Extension-72c-1mt; (A total of 35 cycles); No final extension needed. The PCR products were then run on agarose gel and stained with ethidium bromide and visualized under UV-Gel doc system. Positive controls  Ethical Justification. The blood sample used in the study is withdrawn as a part of routine diagnostic work-up of the patient and no additional sample pricks were performed. Prior informed consent was taken from all patients/guardians before withdrawl of sample.
Results. The multiplex RT-PCR reactions were carried out in a total of 116 AML cases. Of these, 96 (83%) were adult and 20 (17%) pediatric cases. The median age in AML cases for adults was 42 years (range 14-40 years) and for pediatric was 6 years (range0.6-12 years). The male to female ratio in adult AML cases was 1:1 and pediatric 2:1. 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. The percentage positivity for the common fusion transcripts in adult and pediatric cases is outlined in Table 2. Figure 1 shows the positive band positions for the different chimeric transcripts in AML cases and Figure 2 highlights the positivity for bcr1 isoform of PML-RARA in the separately run multiplex RT-PCR.
Discussion. In the present study, multiplex RT-PCR was performed for common fusion transcripts of AML1-ETO, PML-RARA and CBFβ-MYH11 as it is a very cost effective screening procedure in a resource constraint setting.  The incidence of the common fusion transcripts in present study was 33.6%. The same is in accordance with other studies from Asian countries. Table 3 and 4 highlights the positivity of common fusion transcripts in various AML both adult and pediatric cases.
AML1-ETO was the most common fusion transcript noted in both adult (14.58%) and pediatric (30%) cases. The incidence of AML1 is higher in our adult AML cases as compared to the study in west. This can be partly explained due to a smaller sample size in our study, more clustering of cases as our being a tertiary referral hospital or due to certain ethnic, genetic and environmental factors, which need to be evaluated in further prospective studies. In all 39 positive cases a retrospective evaluation of morphology was also done to do a morphological-genotypic correlation. A concordance rate of 82% was noted between morphology and genotype in adult cases and 91% in pediatric cases. The classical morphology was able to predict genotype accurately (100%) in inv16 positive cases i.e. AML M4 with Eo on morphology. This was followed by an accuracy of 85% for AML-Eo) and 80% for AML promyelocytes with aeur rods). Lower predictability for AML-ETO and PML-RARA is noted because cases with classical morphology were only considered but it is well described that AML can also show AML-ETO positivity and PML positivity can also be seen in other morphological variants of AML-M3. However there was a better correlation with genotype and morphology in PML RARA cases if immunophenotype findings o and HLA-DR negativity were taken into account (95%).
Disease monitoring by RT done only for PML-RARA cases in our institute, with evaluation at either post induction or post consolidation phase in cases with complete morpholo hematological remission. In present study, follow data on RT-PCR is available for only 4 adult and 1 pediatric case positive for PML Table 3. Shows the incidence of common transcripts in AML in various studies

Study
Number AML cases

1) Present study
2) Olesen et al The incidence of the common fusion transcripts in present study was 33.6%. The same is in accordance with other studies from Asian 1,2 and Western 3,4,5 highlights the positivity of common fusion transcripts in various AML studies in both adult and pediatric cases.
ETO was the most common fusion transcript 58%) and pediatric (30%) The incidence of AML1-ETO and PML-RARA is higher in our adult AML cases as compared to the study in west. This can be partly explained due to a smaller sample size in our study, more clustering of cases as our being a tertiary referral hospital or due to certain ethnic, genetic and environmental factors, which need to be evaluated in further prospective studies. In all 39 positive cases a retrospective evaluation of morphology was also done to do a genotypic correlation. A concordance 82% was noted between morphology and genotype in adult cases and 91% in pediatric cases. The morphology was able to predict genotype accurately (100%) in inv16 positive cases i.e. AML-M4 with Eo on morphology. This was followed by an -ETO cases (AML-M2 with Eo) and 80% for AML-M3 cases (classical promyelocytes with aeur rods). Lower predictability for RARA is noted because cases with classical morphology were only considered but it is well described that AML-M1 cases on morphology ETO positivity and PML-RARA positivity can also be seen in other morphological M3. However there was a better correlation with genotype and morphology in PML-RARA cases if immunophenotype findings of CD34 DR negativity were taken into account Disease monitoring by RT-PCR is presently being RARA cases in our institute, with evaluation at either post induction or post consolidation phase in cases with complete morphological and hematological remission. In present study, follow-up PCR is available for only 4 adult and 1 pediatric case positive for PML-RARA. Of these all Shows the incidence of common transcripts in AML in  Various studies 6-8 have highlighted comparable or slightly higher sensitivity of RT-PCR as compared to cytogenetic/FISH analysis in detecting recurrent fusion transcripts in AML. It is impressed upon in these studies that routine cytogenetic analysis should be done in all AML cases to identify genetic abnormalities in AML and RT-PCR should always supplement it to define variant/cryptic translocations or detect fusion transcripts in cases with poor quality metaphases on cytogenetics. However we could not carry out detailed cytogenetic analysis or FISH in any of our AML cases due to technical limitations and cost factor and hence a comparison could not be highlighted between these techniques in our study.
Take Home Message. Multiplex RT-PCR is not only a sensitive technique than conventional cytogenetics or but also is easy to perform and less time consuming.
Screening for the common fusion transcripts should be routinely carried out in all AML cases as it guides the clinician in taking important therapeutic decisions and provides prognostic information.