Introduction

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder resulting from neoplastic transformation of haemopoietic stem cells that can lead to increase of myeloid series in peripheral blood and myeloid hyperplasia in bone marrow. The most common adult leukemia in India is CML with annual incidence ranging from 0.8-2.2/100,000 population in males and 0.6-1.6/100,000 population in females [1]. The discovery of the first chromosomal abnormality in CML was done by Nowell and Hungerford in 1960 in Philadelphia and the deletion of one of the shortened chromosome of chromosomes 22 pair was named as ‘Philadelphia chromosome’ to honor the city [2]. The presence of Philadelphia chromosome (Ph) differentiates the chronic myeloid leukaemia from other chronic myeloproliferative disorders [3].

Philadelphia chromosome arising from the reciprocal translocation between long arm of chromosomes 9 and 22 that fuses the ABL (Abelson Murine leukemia Virus) oncogene on chromosome 9 to the BCR (breakpoint cluster region) gene on chromosome 22. This results in the formation of chimeric fusion gene BCR-ABL and this translocation - t(9;22)(q34;q11.2) was identified with advancement of banding techniques which became the diagnostic hallmark of CML. Conventional cytogenetics or karyotyping is the standard method to detect chromosomal aberration. In addition to Ph chromosome, chromosomal abnormalities like trisomy 8 and isochromosome 17 also can be identified in CML cases by using chromosomal analysis [3].

Fluorescence in situ hybridization (FISH) is a molecular cytogenetics technique, becoming a vital part of clinical practice in the workup of patients with hematological malignancies. FISH can be used to investigate both proliferating (metaphase cells) and non-proliferating (interphase nuclei) cells by detecting the location of specific nucleic acid sequences, using fluorescently labeled specific DNA probes to the target sample. FISH has been used as the most reliable method to identify the BCR/ABL gene at the time of diagnosis [3].

Materials and Methods

Subject

A 52 year old female presented with fatigue and fever of one month duration was admitted in K. S. Hegde Charitable hospital, Deralakatte, Mangalore. On examination, she had pallor and splenomegaly 4cms below the costal margin. The blood investigations revealed total WBC count of 2.5×10⁹/L and hemoglobin level of 9gms/dl. Peripheral blood smear confirmed the diagnosis of CML. (Differential count -14 nRBCs/100 WBC, Myeloblast - 11%, Promyelocytes - 7%, Myelocytes - 21%, Metamyelocytes - 13%, Band forms - 12%, Neutrophils - 23%, Eosinophils - 8%, Basophils - 5%.) Peripheral blood smear picture of the patient is shown in Figure 1 & 2.

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Figure 1 & 2:

Peripheral blood smear - Leishman's stain x400 and x 1000 Observe increased WBC count and the presence of increased myeloid series cells.

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Owing to her high WBC count, 3ml of peripheral blood was collected in sodium heparin vacutainer tube and sent for karyotyping and Fluorescence in situ hybridization (FISH) to Diagnostic Centre for Cytogenetics and Molecular genetics.

Conventional cytogenetic analysis

Direct and 24 hour cultures were set up with peripheral blood using RPMI 1640 medium supplemented with 20% of fetal bovine serum. GTG banding was done using standard cytogenetic protocol. Metaphases were analyzed from each culture, captured using the Olympus BX53 microscope and karyotyped with the ‘GENASIS’ software. Ph chromosome positivity was expressed in all 20 metaphases analyzed and the karyotype was reported according to An International System for Human Cytogenetic Nomenclature (ISCN) 2013.

Fluorescence in situ hybridization (FISH) Analysis

FISH analysis was carried out on interphase cells and metaphases of the fixed cell pellet using BCR/ABL Translocation, Dual fusion probe (Cytocell, UK). The BCR/ABL probe set comprises a BCR probe mix directly labeled in green (Spectrum Green) spanning at 22q11.22-q11-23 and the ABL1 probe directly labeled in red (Spectrum Orange) spanning at 9q34.11-q34.12. Cell pellet was dropped on the slide and 10μl of probe was applied to the target area. The sample and probe were co-denatured and hybridized using the ThermoBrite Denaturation /Hybridization System.

The ThermoBrite unit was programmed to allow 5 minutes of denaturation at 73°C to transform the double strands DNA into a single strand, followed by overnight hybridization at 37°C for the probe to hybridize with the target cells and bind to it. Next day, the slide was washed with 0.4X SSC/0.1% NP-40 at 72°C for 2 minutes, followed by a wash in 2X SSC/0.1% NP-40 at room temperature for 1 minute to remove any of the probes that did not bind to the cells. The slide was air dried in the dark, then added 10μl of DAPI (4, 6-diamidino-2-phenylindole) as a counter stain.

The FISH signals were visualized using an Olympus BX53 fluorescence microscope equipped with appropriate filters. Interphase nuclei and available metaphases were scored and the signals were captured using a CCD camera attached to a FISH View image acquisition and analysis system for FISH (GENASIS, Applied Spectral Imaging, Germany).

Results

Cytogenetic analysis of a metaphase showing 46, XX, t(9; 22)(q34; q11. 2) karyotype, which results in the Philadelphia chromosome is shown in Figure 3. FISH analysis of normal interphase nuclei will have a simple 2R, 2G signal pattern.

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Figure 3:

GTG-banded karyogram of the patient showing 46, XX, t(9; 22)(q34; q11.2)

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As a result of the Philadelphia chromosome, ABL1 (Abelson) proto-oncogene and the BCR (Break point Cluster Region) gene fuse, giving rise to the BCR/ABL1 fusion gene showed 1 green, 1 red and 2 yellow fusion signals (1G, 1R, 2Y) in all the cells analysed (Figure 4 & 5).

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Figure 4 & 5:

Representative interphase cell and metaphase showing BCR/ABL fusion as yellow signals, which represents overlapping of BCR (green) and ABL (red) signals. The other red signal represents ABL on chromosome 9 and green represents BCR on chromosome 22.

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Discussion

Ph chromosome is present in 95% of CML cases and also present in 5 % of children and 20% of adults with acute lymphoblastic leukemia (ALL) [3]. Conventional cytogenetics is considered as the diagnostic hallmark for identification of Ph positive metaphases in CML cases when it is present in more than 30% cells. This method has its own limitations like increased turn around time, labour intensive procedure and analysis dependent on high numbers of viable dividing cells and well spread metaphases. Reena et al studied the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of conventional karyotyping, reported as 85%, 100%, 100% and 71% respectively. The accuracy for conventional karyotyping method was 88% [3]. In the present study, conventional cytogenetics was done with 20 metaphase spreads and the Philadelphia chromosome (Ph+) was seen in all well spread metaphases. Good metaphase spreads are difficult to get and may consume extra time for analysis. However, conventional cytogenetics has its strength of detecting additional nonrandom chromosomal aberrations within the 20 spreads [4].

The quantification of BCR-ABL positive cells by FISH facilitates the monitoring of disease response to therapy [2]. The accuracy of FISH in the analysis of BCR-ABL rearrangements of CML patients has been demonstrated by several recent studies [5, 6, 7]. Even 1-2% of positive cells can be picked up due to the sensitivity of FISH test [5]. FISH was carried out in this patient with 200 interphase nuclei and available metaphases. The presence of Ph chromosome was confirmed and proved the accuracy of FISH on peripheral blood which was 100%. This technique has many advantages, which include analysis of large number of cells, speed of reporting within 24 hours of the sample received in the laboratory, which may be useful in urgent diagnostic situations. It can be performed with fixed cell pellets left over from cytogenetic study of bone marrow or peripheral blood and paraffin embedded tissues [3].

Le Gouill et al studied the correlation between FISH and cytogenetics on bone marrow and got highly correlated results, then they moved on to the analysis of peripheral blood samples where comparable results were obtained, with an excellent correlation (r = .97) [6]. Earlier reports have demonstrated that FISH technique was able to detect all types of BCR-ABL fusions in CML with masked or variant Ph, which is not apparent with the conventional karyotyping [6, 8]. Lim et al observed additional karyotypic changes like supernumerary Ph or deletions of 9q and/or 22q in 28.1% of Ph positive patients by interphase FISH [8]. The detection of BCR-ABL cells by Interphase FISH correlates better with molecular response, the exact interpretation of FISH in conjunction with chromosomal analysis is the best practice, to avoid the possibility of misdiagnosis [8, 9].

Patel et al observed significantly reduced disease free survival in patients with deletion in ABL, BCR or ABL, BCR on derivative 9 as compared to non-deleted derivative 9 and their findings suggest that the gene segments around the breakpoint are deleted in heterogeneous patterns when the translocation occurs and the genes located near the breakpoint may also play a role in the delayed tumor progression [10]. Thus, FISH technique gives information about the phases of CML and useful at diagnosis after BMT (Bone Marrow transplantation) for detection of residual diseases.

Conclusion

Chromosomal analysis and FISH both have their advantages and should ideally be carried out together in order to make a correct diagnosis and predict/monitor response to newer molecular targeted treatment modalities like imatinib mesylate. FISH can be done in peripheral blood when the WBC count is high which can result in increase in percentage of Ph positive cells and also failed cases of conventional cytogenetic analysis to quantify disease in CML. FISH on peripheral blood exhibited a high degree of correlation with bone marrow, as a rapid, accurate and relatively non-invasive method.