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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 43  |  Issue : 3  |  Page : 103-108

Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia


1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Internal Medicine/Hematology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission18-Nov-2017
Date of Acceptance13-Jun-2018
Date of Web Publication3-Dec-2018

Correspondence Address:
Rania S Swelem
Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, 21131
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_50_17

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  Abstract 


Background Numerous recurrent chromosomal aberrations have been identified in acute myeloid leukemia (AML), and their detection has become essential for accurate diagnosis, classification, and prognosis of the disease. Fluorescence in-situ hybridization (FISH) provides a powerful technique complementary and even alternative to chromosome banding studies for the identification of selected chromosome aberrations.
Aim Evaluation of the multiprobe FISH panel in the detection of common cytogenetic abnormalities in AML and to investigate their association with clinical diagnosis, chemotherapy, and prognosis.
Patients and methods This study was conducted on 20 newly diagnosed AML patients. All patients were subjected to full history taking, clinical examination, laboratory investigations including complete blood count, bone marrow aspiration, immunophenotyping, and interphase FISH using cytocell multiprobe AML/myelodysplastic syndrome panel designed to detect AML/Eight-twenty-one (ETO), Promyelocytic leukaemia- Retinoic acid receptor alpha (PML-RARA), and core-binding factor beta/Myosin, heavy chain 11, smooth muscle (CBFβ/MYH11) translocations, mixed-lineage leukaemia (MLL) break apart, P53, 5q, 7q, and 20q deletions.
Results Interphase FISH analysis showed 5q deletion in 8/20 (40%) cases, positive PML/RARA in 3/20 (15%) cases p53 deletion in 15/20 (75%) cases, positive AML1/ETO in 1/20 (5%) cases, no MLL break apart cases (0%), 7q deletion in 4/20 (20%) cases, positive CBFβ/MYH11 fusion gene in 4/20 (20%) cases, 20q deletion in 9/20 (45%) cases, and trisomy 8 in 7/20 (35%) cases. There was a statistically significant relationship between 5q deletion and prognosis (P=0.028).
Conclusion Multiprobe FISH is more cost effective and time effective compared with traditional FISH. It is an efficient technique for the detection of cytogenetic aberrations AML, providing critical information for diagnosis and prognosis, and for monitoring the course of the disease.

Keywords: acute myelogenous leukemia, chromosomal aberrations, fluorescence in-situ hybridization, multiprobe panel, prognosis


How to cite this article:
Sorour AF, Nafea DA, Swelem RS, Soliman EM. Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia. Egypt J Haematol 2018;43:103-8

How to cite this URL:
Sorour AF, Nafea DA, Swelem RS, Soliman EM. Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia. Egypt J Haematol [serial online] 2018 [cited 2018 Dec 16];43:103-8. Available from: http://www.ehj.eg.net/text.asp?2018/43/3/103/246779




  Introduction Top


Acute myelogenous leukemia (AML) is a heterogeneous group of disease characterized by uncontrolled proliferation of myeloid progenitor cells that gradually replace normal hematopoiesis in the bone marrow (BM) [1]. The genetic changes arising in the neoplastic clone lead to cascades of molecular events that cause abnormal proliferation, aberrant differentiation, and inhibition of normal hematopoiesis by the malignant cells [2],[3],[4]. Cytogenetic aberrations are found in about 60% of AML cases [5].

Characterization of transforming genetic events is increasingly important in establishing diagnosis, defining prognosis, and planning therapy in AML [6]. The current challenge is to understand the molecular mechanisms of AML and design leukemia-specific treatments effective in chemotherapy-resistant disease and applicable to older patients [2].

Conventional G-banding analysis (G-banding) is the most commonly used method to detect cytogenetic abnormalities. However, the detection of clonal chromosomal abnormalities by G-banding is often unsuccessful; subtle or submicroscopic (i.e. cryptic) rearrangements affecting regions smaller than a chromosomal band cannot be detected by G-banding. Fluorescence in-situ hybridization (FISH) may be used to supplement G-banding, but FISH is restricted to the defined chromosome regions of the probes used. Multiprobe FISH panels are designed to detect up to eight different FISH probes on a single slide. Multiple FISH probes can be hybridized on the same slide in a spatially separated manner, allowing rapid analysis of cytogenetic aberrations in a single hybridization experiment. Thus, multiprobe FISH assay can be used effectively in AML [7].


  Aim Top


The aim of this study was to evaluate the value of multiprobe FISH panel in the detection of common cytogenetic abnormalities in AML and to investigate their association with clinical diagnosis, chemotherapy, and prognosis.


  Patients and methods Top


The study was conducted on 20 consecutive newly diagnosed AML patients attending the Hematology Unit, Faculty of Medicine, Alexandria University Hospital in the period from February 2015 till April 2016.

The diagnosis of AML was based on WHO 2008 diagnostic criteria using the standard methods including morphological, immunophenotypic, and cytogenetic evaluation. Patients suffering from any other hematological malignancies or any other malignancies were excluded from our study.

A written informed consent was obtained from all participants enrolled in this study and all procedures performed in our study involving human participants were in accordance with the ethical standards of our institution and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

All patients in the study were subjected to full history taking, complete clinical examination and laboratory investigations including complete blood count performed on a five-part differential automated cell counter Siemens ADVIA 2120i (Siemens Healthineers, Forchheim, Germany) hematology analyzer. Peripheral blood (PB) smears were spread, air-dried, stained by Leishman’s stain, and microscopically examined to assess the peripheral differential blood cell counts and morphology and the blast percentage [8],[9]. BM aspirate examination was done for all patients using Klima needle. Aspiration was done from the posterior superior iliac spine or sternum; air-dried films from BM aspirate were stained by Leishman’s stain for morphological examination [10]. Immunophenotyping by flowcytometry: immunophenotyping of the leukemic blast cells was performed on PB or BM samples in K2EDTA tubes. Briefly, 10 µl of the cluster of differentiation (CD) was added to 100 µl of whole EDTA blood, mixed well, and incubated for 10 min at room temperature. The cells were then washed twice with PBS, 2 ml lysing solution were added, mixed, and left for 10 min in the dark and then washed twice with PBS. After the last wash, the cells were analyzed by BD FACSCaliber flow cytometer (serial number E34297300591; BD BiosciencesSan Jose, California, USA) equipped with the CellQuest software (BD Bioscience, San Jose, California, USA). During analysis a gate was set around the required blast population. The cut-off point of positivity was considered when more than 20% of the cells are stained with an antibody in excess of the background fluorescence in the negative controls. An extra step of fixation and permeabilization was done when analyzing intracellular antigens.

The following panel of monoclonal antibodies for the diagnosis of acute leukemia was applied [11]. primary panel: CD2-PE (Immunostep, Salamanco, Spain), CD5-PE (BD), CD7-FITC (BD), CD10-FITC (BD), CD19-RPE (BD), CD14-FITC (BD), CD13-PE (BD), CD33-PE (Immunostep), HLA-Dr-FITC (BD), CD34-FITC (BD), and CD45-FITC (BD). Confirmatory antibodies: Cyt CD22-FITC (BD), CytIgM-PE (BD), Cyt CD3-FITC (Immunostep), and Cyt anti-MPO-FITC (Immunostep).

Interphase fluorescent in-situ hybridization

The Cytocell Multiprobe AML/myelodysplastic syndrome (MDS) panel (UKPI027/CE v005; Cytocell Technologies Ltd., Cambridge, England) designed to detect up to eight different FISH probes was used on a single slide in a single hybridization experiment which was for the AML/Eight-twenty-one (ETO) translocation gene, promyelocytic leukaemia- retinoic acid receptor alpha (PML-RARA) translocation gene, core-binding factor beta/Myosin, heavy chain 11, smooth muscle (CBFβ/MYH11) translocation gene, mixed-lineage leukaemia (MLL) break apart, P53 deletion, Del(5q), Del(7q), Del(20q) [12].

Sample was peripheral blood or bone marrow aspirate on Na heparin

Blood was transferred to a 15 ml conical centrifuge tube; they were centrifuged at 1500 rpm for 10 min; the supernatant was carefully removed; 10 ml 0.075 M hypotonic KCl were then added (prewarmed at 37°C), mixed well, and left to stand for 20 min at 37°C. At the end of the 20 min, the tubes were centrifuged, the supernatant discarded, and then 10 drops of freshly prepared fixative were added in a dropwise manner with good mixing in between. The tubes were then centrifuged at 1500 rpm for 10 min, the supernatant was discarded and the cell pellet was resuspended in 1 ml freshly prepared fixative added dropwise with shaking. Then, the remaining fixative (9 ml) was slowly added and the suspension was mixed. The tubes were then centrifuged at 1500 rpm for 10 min; the supernatant was discarded; and the cell pellet was resuspended again in 10 ml fixative with mixing. The washing steps with the fixative were repeated two more times, until the cell pellet appeared clean and fixative was clear. After the last supernatant was discarded, enough fixative was added to obtain a slightly cloudy suspension. Slides were then made or the suspension kept in the refrigerator at 2–8°C till slide preparation was done.

In brief, the template slide was soaked for 2 min in 100% methanol and then dried; 4 µl of the cell suspension was added to each of the eight areas of the slide in a sequence of alternating squares. The slides were then dipped in 2×side scatter (SSC) for 2 min at room temperature then dehydrated in ascending grades of ethanol (70, 90, and 100%) for 2 min each, left to air dry then warmed up at 37°C on hot plate. A measure of 2 µl of the prewarmed hybridization solution was then added to each square of the eight areas.

The template slide was then carefully inverted on the prewarmed device and placed at 37°C in the Hybrite (Vysis serial number 114650) for 10 min followed by denaturation at 75°C for 2 min; finally the slide device sandwich was placed in the prewarmed chromophobe hybridization chamber which was left to float at 37±1°C in the water bath overnight (or in the Hybrite). The device was then removed from the slide and the slide was then washed, DAPI/antifade applied, cover slip applied and finally examined by the fluorescence microscope (Olympus Microscope BX51/61, Olympus, Japan) and by using CytoVision Applied Imaging (Newcastle upon Tyne, England).

Statistical analysis of the data

Using IBM SPSS software package version 20.0 the qualitative data were described using number and percentage (SPSS Inc., Chicago, Illinois, USA). Comparison between different groups regarding categorical variables was tested using the χ2-test. When more than 20% of the cells have expected a count of less than 5, correction for χ2 was conducted using Fisher’s exact test or Monte Carlo correction [13],[14].

Quantitative data were described using mean and SD for normally distributed data, whereas abnormally distributed data were expressed using median, minimum, and maximum.

For abnormally distributed data, Mann–Whitney test was used to analyze two independent populations.

Results interpretation

The following cut-off values were used in our study (these cut-off values were determined in our laboratory and are method dependent): AML/ETO translocation gene 3%, PML-RARA translocation gene 3%, CBFβ/MYH11 translocation gene 3%, MLL break apart 3%, P53 deletion 6%, Del(5q) 6%, Del(7q) 4%, and Del(20q) 4%. A total of 500 cells were counted per probe by two doctors to determine the results of the cases.


  Results Top


Patients included in the study were 11 men and nine women, the age ranged between 20 and 76 years with a mean of 49.0±14.10; the median was 49 years. We had two (10%) cases of M0, two (10%) cases of M1, three (15%) cases of M2, four (20%) cases of M3, only one (5%) case of M4, four (20%) cases of M4EO, and four (20%) cases of M5. Patients characteristics are described in detail in [Table 1]. Interphase FISH analysis was successfully performed on the 20 BM or PB samples. Positive Tp53 deletion in 15/20 (75%) cases, PML/RARA translocation was positive in 3/20 (15%) cases, deletion 5q was positive in 8/20 (40%) cases, deletion 7q was positive in 4/20 (20%) cases, deletion 20q was positive in nine (45%) cases, and CBFβ/MYH11 fusion gene was positive in 4/20 (20%) cases, Trisomy 8 was positive in 7/20 (35%) cases, only one (5%) case was positive for AML1/ETO, whereas no MLL dual fusion positive cases were found ([Figure 1]).
Table 1 Distribution of the cases according to demographic, clinical characteristics, and laboratory findings of the acute myeloid leukemia patients

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Figure 1 Representative interphase fluorescence in-situ hybridization images showing: (1) 5q deletion, (2) PML/RARA, (3) p53 deletion, (4) AML (RUNX1)/ETO, (5) MLL rearrangement, (6) monosomy 7, (7)t(16;16), and (8) monosomy 20 in areas 1–8, respectively.

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There was a statistically significant difference between TP53 deletion and BM blasts (P=0.026) and white blood cells (WBC) counts (P=0.027), between PML/RARA translocation and BM blasts (P=0.044), WBC counts (P=0.001) and prognosis (P=0.049), between deletion 5q and outcome (P=0.026), between 7q deletion and WBC counts (P=0.001) and PB blasts (P=0.006), between CBFβ/MYH11 fusion gene and age (P=0.005), and finally between Trisomy 8 and outcome (P=0.009), BM blasts (P=0.021) and lymphadenopathy (P=0.051), while there was no statistically significant difference found between 20q deletion and age, sex, clinical symptoms, outcome, prognosis, organomegaly, peripheral blasts, BM blasts, and complete blood count findings.

As regards prognosis, we found that P53 Del was the most sensitive parameter (66.67%) followed by Del 20q and Trisomy 8 (22.22%), whereas CBFβ/MYH11 fusion gene and Del 5q were the least sensitive ones. On the other hand, CBFβ/MYH1 fusion gene and PML/RARA were the most specific parameters (72.73%).

We had only two cases that had no FISH abnormalities regarding our studied probes and both cases went into complete remission. Six cases were only positive for one gene (five TP53 and one Trisomy 8), five of them went into complete remission whereas one case only was refractory for treatment. We had two cases who were positive for two genes together and both died (one case was positive for 20q deletion and 5q deletion, whereas the other one was positive for PML/RARA and Trisomy 8), The remaining 10 cases were positive for more than two genes; eight cases were of bad prognosis (five refractory and three died) whereas only two went into remission.


  Discussion Top


Current management of patients with AML is determined by a number of parameters, including age, performance status, and the cytogenetic/molecular genetic characteristics of the leukemic clone. Together, these factors have an important bearing on treatment strategy, identifying the potential candidates for molecularly targeted therapies and informing decisions on allogeneic transplantation [7].

To our knowledge, there are not many papers that have been published using the Chromophobe Multiprobe AML/MDS panel and its usefulness for detection of the most common genetic rearrangements in AML as well as other structural and numerical abberations.

Valencia et al. [7], in a study conducted on 80 patients using both cytogenetics and multiprobe FISH, identified seven (9%) patients with good-risk cytogenetics: four patients with inv(16)(p13q22), two patients with t(8;21)(q22;q22), and one with t(15;17)(q22;q21). Five rearrangements were detected by both methods, whereas one inv(16) and one t(15;17) were only detected by FISH. With regard to patients with intermediate risk cytogenetics, they found complete concordance between both methods in all 33 (41%) patients with normal karyotype except for the aforementioned cryptic t(15;17). Nineteen (24%) additional patients of the intermediate category had other miscellaneous structural or numerical defects. The multiprobe AML panel was useful in the characterization of chromosomal aberrations in 15 (19%) patients with adverse risk cytogenetics. It showed a good correlation with conventional karyotype in all cases except one, who had a cryptic deletion of the CBFβ allele and was detected only by FISH. FISH was ineffective in detecting aberrations in six (7.5%) cases that involved chromosome regions not represented in the multiprobe panel. However, the panel helped to identify aberrations in nine (11%) patients without metaphases (seven cases) or with noninterpretive chromosomes (two cases): three MLL rearrangements, two monosomy 7, one of them also with del (5q), and one with inv(16)(p13q22); no alterations were found in the remaining three cases [7].

Thus, similar to our conclusion, they reported the usefulness of the multiprobe FISH in AML, showing that this assay was very useful in the detection of an inv(16)(p13q22), a cryptic t(15;17)(q22;q21), and a cryptic deletion of the CBFβ.

Valencia et al. [7], reported that the multiprobe FISH panel has some disadvantages which should be considered.

Additional single probes were sometimes needed to complete the diagnosis in some cases. Regarding the interpretation of the results, they obtained optimal hybridization signals generally, but occasionally they found a few cases with cross-hybridization interfering between different probes at the boundaries of the eight spots. Although these artifacts were easy to detect, detailed molecular knowledge and skills for interpretation were required, to avoid misleading results. Finally, as this commercial multiprobe kit examines the loci of 10 different chromosomes, it is unable to detect nonrecurrent translocations, as well as numerical and structural defects that involve chromosomes not included in the panel [7].

However, we did not face technical problems while working on this panel. No cross-hybridization occurred on any of the cases. We did not need any additional probes to diagnose any of the cases, unless signal failure could happen at one or more of the loci due to technical error.

In another research that used the multiprobe panel, Kim et al. [15] compared multiprobe FISH results with those of G-banding in a study conducted on 30 acute leukemia patients, including 15 AML and 15 ALL cases; multiprobe FISH identified additional genetic aberrations in six of 30 cases (two and four in AML and ALL, respectively) including AML1/ETO fusion, MLL rearrangement, and CBFβ/MYH fusion. Multiprobe FISH additionally identified TP53 deletion in AML cases; thus, it can reliably detect TP53 deletion and it was found in 4.5–9% of AML cases [15].


  Conclusion Top


Multiprobe FISH assay is an efficient technique for the detection of cytogenetic aberrations in AML, providing critical information for diagnosis and prognosis, and for monitoring the course of the disease.

We found that TP53 deletion was the most frequent abnormality detected which made it the most sensitive parameter predicting prognosis, whereas AML/ETO and MLL rearrangement were the least frequent ones.

The following points can be recommended:
  1. We recommend the use of the multiprobe AML/MDS panel for detection of cytogenetic aberrations in AML and MDS for diagnosis, prognosis, and monitoring of the disease as it is more cost and time effective compared with traditional FISH and in detection of MRD in AML patients positive for the marker at the diagnosis should be investigated.
  2. It is preferred to do cytogenetics for all AML cases for scanning and risk stratification, as FISH, PCR, and conventional cytogenetics results need to be considered together, as all these methodologies can provide additional information for diagnosis, genetic risk assignment, and follow-up of patients with AML.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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