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VOLUME 48 (2) : DEC 1999


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Dr. Patnaik V.V. Gopichand

Appearance of fast blue in the ventral spinal cord following section of the sciatic nerve in rats.
*Sabita Mishra, **Madhur Gupta, ***P. Sengupta

Organisation, Behaviour and Significance of the Changing Pattern of Neural Elements in the Trigeminal Ganglion during Development and Aging : An Investigation in the Chick
*A.G. Pillay and Vennila, G.D.

Potentiation of Teratogenicity by Acute Hypoxia in Animal Model and Its Hypothetical Clinical Implications
Nandita Datta* & Gajendra Singh**

Assessment of Philadelphia Chromosome status in Chronic Myelogenous Leukemia (CML) Patients Using Cytogenetic and Molecular Cytogenetic Methods
V Jobanputra, TA Sivakumaran, K Kucheria

Incidence of Subsuperior Bronchus - Morphological and Bronchographic Study.
*Patnaik V.V.G. & l Saha J.C.

Letters to the Editor
Kalsey, G. Patnaik, V.V.G. & Singla, R

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J Anat. Soc. India 48(2) 90-98 - (1999)
Assessment of Philadelphia Chromosome status in Chronic Myelogenous Leukemia (CML) Patients Using Cytogenetic and Molecular Cytogenetic Methods

V Jobanputra, TA Sivakumaran, K Kucheria

Abstract : Chronic myelogenous leukemia (CML) is a clonal bone marrow disease characterized by neoplastic overproduction of granulocytes. Cytogenetic characteristics of CML is the presence of the Philadelphia (Ph) chromosome which involves rearrangement of BCR-ABL g

Keywords : Ph Chromosome, CML, FISH, BCR-ABL Probe

Introduction

Chronic Myelogenous Leukemia (CML) is a clonal myeloprolifrative disorder. It results from the neoplastic transformation of the primitive hemopoetic stem cell. CML accounts for about 7 ? 15% of all leukemia�fs in adults with approximately 1?1.5 cases per 100,000 population. (Morrison et al., 1994)

The course of the disease demonstrates a multistep neoplastic progression. The first or the chronic phase (CP) is relatively indolent with a median duration of 3.5 years but there is a wide range of variation. The chronic phase leads to an accelerated phase, when differentiation of the myeloid fails and disease gradually becomes refractory to treatment. The terminal phase is marked by heavy increase in the proportion of myeloid progenitors in the peripheral blood which predominate in the circulation. The terminal phase is an acute, which is known as blast crisis. It can be lymphoid or myeloid in nature. (Champlin and Glode, 1985) Acute phase of the disease is fatal.

The standard diagnostic criteria are leukocytosis, increased myelopoiesis, basopilia, and/or eosinophilia, decreased leukocyte alkaline phosphatase (LAP) and hepatosplenomegaly. The bone marrow is hypercellular, with an increased myeloid erythroid ratio. The common symptoms in chronic phase include fatigue, weakness and pallor related to anemia, and symptoms
related to splenomegaly.

The cytogenetic hallmark of CML is Philadelphia (Ph) chromosome, the small chromosome 22, which is present in over 90% of cases. This results from reciprocal translocation of the chromosome no. 9 and no. 22, t(9;22) (q34; q11). The formation of the Ph chromosome at the molecular level results from fusion of Abelson (ABL) proto-oncogene from chromosome 9q34 with the break point cluster region (BCR) at chromosome 22q11 to give a hybrid BCR-ABL gene and expression of the fusion protein, BCR-ABL p210 with a molecular weight of 210 KD. (Konopka, Watanabe & Singer 1985; Ben Neriah, Daley & Mes-Masson. 1986) The BCR-ABl p210 protein has augmented tyrosine kinase activity and is thought to be important in the pathogenesis of CML. (Kloetzer, Kurarock & Smith 1985; Clark, Laughlin & Crist. 1987).

The Ph chromosome is the sole genetic abnormality during the CP but variants of it involving other chromosomes are also seen. Such variants do not show any deviation from clinical course. The advanced stage shows additional genetic abnormality in about 80% cases which are probably a part of the neoplastic progression. Identification of the Ph chromosome derived from t(9;22) (q34;q11) in these cases using conventional cytogenetic analysis requires a well spread metaphase chromosome preparation.

There are cases which show clinical featurs of CML but do not show a Ph chromosome by cytogenetics. These cases either have submicroscopic translocation, where the underlying molecular event has taken place, or a low mosaiscism is present. Recently introduced Flourescence In Situ Hybridization (FISH) technique can be used on poorly spread metaphases as well as on interphase nuclei to identify the Ph chromosome or BCR-ABL fusion gene at the molecular level.

It depends on the hybridization of labelled DNA probes to their complementary sequences on metaphase spreads or interphase nuclei. A series of optical filters are used to capture the image of each fluorophor separately using a charged-coupled device (CCD). Then a computer program is used to merge the separate CCD camera images into a composite image, in which each chromosome or chromosomal region is assigned a distinct color based on its fluorophor composition.

Conventional chemotherepy of CML includes hydroxyurea and busulfan as anti-CML agents. These provide excellent disease control with minimal toxicity, are inexpensive and are administered orally. But none of these can stop or delay the eventual clinical progression. Cytogenetic response apart from hematological remision is rare. Interferon as a bio-therapy has given better prognosis leading to cytogenetic remission and prolonged survival. It is only the bone marrow transplantation which has altered the advancement towards the final phase, though it can be applied to a limited group of patients. Thus, an optimal therapy for CML is still not final.

The present study was aimed to detect Ph chromsome using conventional chromo-somal analysis, and to further analyze these cases using molecular cytogenetic method of FISH in order to compare the two methods and validate the results. We have used FISH technique on metaphase chromosome preparations and interphase nuclei obtained from bone marrow aspirates from CML patients in order to assess the diagnostic value of this molecular cytogenetic method.

Patients

Twenty three patients (13 females and 10 males) aged 16-60 years were studied. At the time of study all patients were in chronic phase. Of these five patients were studied at diagnosis (untreated ? 300/96, 323/96, 324/96, 334/96 and 335/96) and rest 18 were on treatment (Table 1).

Cytogenetic Analysis

Metaphase chromosome preparations were made from bone marrow samples using standard techniques with slight modifications. These preparations were then subjected to G-banding. A part of the same cell suspension was used for FISH analysis of metaphase spreads and interphase nuclei.

Bone marrow preparations : About 0.5ml of bone marrow aspirate was collected in a sterile centrifuge tube containig 5ml of RPMI 1640 medium with 100 U/ml heparin. The samples were centrifuged at 100 rpm for 10 minutes. The supernatant was discarded and the cell pellet was distributed into two culture vials (4-5 drops each), each containing 5ml of RPMI 1640 medium supplemented with 20% fetal bovine serum. For direct preparations 0.2 mg/ml colcemid was added and incubated for 2 hours at 37�‹C. This was followed by hypotonic treatment (0�~56% K Cl) and fixation. The 24 hours culture was also incubated at 37�‹C and harvested using the same protocol.

G-banding : The G-banding technique of Seabright (1971) was used for microscopic analysis of metaphases. Four to five days old slides were treated in aqueous trypsin solution (0.05%) for 15-20 seconds. The slides were then briefly rinsed in normal saline and stained in 2% aqueous Giemsa staining solution for 5-7 minutes. The slides were rinsed in distilled water and allowed to air dry. Slides usually at 300-400 band level were used for microscopic scanning and karyotyping.

Microscopy and Image analysis : About 25-30 well spread metaphases were analyzed on the light microscope (Zeiss, Germany). At least 5 G-banded metaphases were karyotyped on image analyzer ? Quips Karyotping software (Vysis, Germany) connected to the microscope. This allowed rapid karyotypic analysis. Two metaphase spreads were photograhed from each case using a slow speed, 25 ASA black and white film (Agfa Copex) for conventional karyotyping.

FISH Analysis

FISH analysis was performed on metaphase spreads and interphase nuclei, using locus specific probes for BCR-ABL gene (Philadelphia chromosome).

Slide preparation : Fixed cell suspension was dropped on to clean, chilled slides and air dried. The slides were observed under a phase contrast microscope and the area with highest cell density, preferably with good number of metaphases was marked using a diamond pencil.

Probes : Commercially available FISH probes (from Vysis, Germany) were used for the study. The probe for the BCR-ABL gene was specific for the BCR locus (22q11.2) labelled with spectrum green and the ABL locus (9q34) labelled with spectrum orange fluorophor.

Denaturation and Hybridization : Denaturation and hybridization of the specimen DNA and the probe was performed on Hybrite, denaturation/hybridization system for FISH (Vysis, Germany) according to the manufacturer�fs instructions. Briefly 10ml of the probe was applied on a hybridization area (marked using a diamond pencil), a coverslip was placed and sealed using rubber cement. Denaturation of the probe and the target DNA was carried out for 2 minutes at 73�‹C �} 1�‹C. Hybridization was carried out at 37�‹C overnight in a humidified box. Next day the slides were washed in 0.3% NP-40 in 0.4�LSSC at 73�‹C for 2 minutes and in 00.1% NP-40 in 0.4�LSSC at room temperature for 1 minute. The slides were air dried and 10ml of counterstain DAPI was applied, covered with a coverslip and sealed.

Microscopy and Image analysis : The slides were examined under a Zeiss Axiophot microscope using a triple band pass filter set (DAPI, FITC and Texas red). Images were captured using a CCD camera attached to the microscope and image analysis was performed using Quips Smart Capture FISH Imaging Software (Vysis).

Scoring criteria for FISH results :

Metaphase Spreads : The ABL (orange) signals are located on both chromosomes 9 at the q34 region whereas BCR (green) are located on both chromosomes 22 in the BCR-ABL negative metaphases. In case of the BCR-ABL rearrangement one of the chromosome 9 has the ABL signal, and one of the 22 has only the green signal, an orange-green doublet or a yellow signal can be located on the other chromosome 22, i.e. the Ph chromosome.

Interphase nuclei : In normal interphase nuclei four separate fluorescent signals i.e. two green and two red are seen. In Ph+ve nuclei, however, there are two separate signals which represent the two normal chromosomes, and an orange-green doublet or a yellow signal corresponding to BCR-ABL fusion gene on chromosome 22.

In the present study a minimum of 20 metaphase spreads were analyzed for flourescent signals. For interphase FISH analysis 50 interphase nuclei were scored and the number of fluorescent signals in each nuclei were recorded for each case. For interphase FISH, nuclei free from any attached cytoplasm or cellular membranes showing 1, 2, 3 or 4 signals were selected for scoring. Only those signals which were well embedded in the nucleus, were included for scoring. Clumped nuclei and the nuclei showing low fluorescence intensity were excluded for analysis.

Results

Cytogenetic analysis of bone marrow aspirates from 23 patients with clinical diagnosis of CML, showed Ph chromosome (Ph +ve) in 15 patients with standard translocation t(9;22) (q34;q11), (Table. I). The abnormal chromosome 22 (Ph chromosome) is seen as a cytogenetically distinct small acrocentric chromosome, and is derived from a reciprocal exchange between chromosome 9 and 22, at bands 9q34.11 and 22q11.2 (Figure 1). Out of these 15 Ph +ve patients, 13 patients were 100% Ph +ve and 2 patients were 70% Ph +ve. Of the rest of the 8 patients, 5 patients were Ph -ve and had a normal chromosome complement and in 3 patients metaphase number was inadequate, therefore cytogenetic results were uninformative.

Preparations from all the 23 patients were subjected to FISH analysis. The presence of BCR-ABL hybrid gene was studied independently on metaphase spreads and/or interphase in these cases. FISH analysis using dual-color FISH probes specific for the BCR (green) and ABL (orange) gene was successful in all the 23 patients.

Table. I : CML Patients Analyzed Using Cytogenetic and Fish Technique

Patients

Therapy

Cytogenetic Analysis

FISH          Analaysis

1(90/94)

BUS, MT

Ph +ve

+ve

2(132/94)

BUS

Ph -ve

-ve

4(434/94)

BUS

Ph -ve

-ve

4(535/94)

HU, IFN

70% Ph +ve

+ve

5(539/94)

HU

Ph -ve

-ve

6(552/94)

BUS, IFN

70% Ph +ve

+ve

7(596/94) 

HU

Ph -ve

+ve

8(49/94)

HU

Ph +ve

+ve

9(55/94) 

HU

Ph +ve

+ve

10(62/94)

HU

Ph +ve

+ve

11(69/94)

BUS

Ph +ve

+ve

12(87/94)

BUS

Ph +ve

+ve

13(90/94) 

BUS, MT

Ph +ve

+ve

14(115/94) 

BUS, MT

Ph +ve

+ve

15(127/94)

-

Ph +ve

+ve

16(132/94)

BUS

Ph -ve

+ve

17(294/96) 

HU

-

+ve

18(300/96) 

-

Ph +ve

+ve

19(304/96) 

HU, IFN

-

-ve

20(323/96)

-

-

+ve

21(324/96) 

-

Ph +ve

+ve

22(334/96)

-

Ph +ve

+ve

23(335/96) 

-

Ph +ve

+ve

BUS = Busulphan
IFN = Interferon
HU = Hydroxyurea
MT = Myeleron therapy

 

Fig.1

 

Fig.2

 

Fig.3

 

On FSH analysis, positive hybridization signals were seen for the BCR-ABL hybrid in the metaphase spreads and interphase nuclei in all the 15 Ph +ve patients. One BCR (green), one ABL (orange) and one BCR-ABL hybrid (yellow) signals were seen in the metaphase spreads as well as on interphase nuclei (Figure 2) of these patients. A few metaphase spreads showed two double hybridization signals, probably as a result of hybridization to both sister chromatids of both homologues.

Of the 5 Ph -ve patients, 4 were negative for BCR-ABL hybrid on FISH analysis. In these 4 patients two BCR (green) and two ABL (orange) signals were seen on metaphase spreads and interphase nuclei (figure 3) whereas one of the Ph -ve patient (596/98) showed positive signals for BCR-ABL hybrid oncogene.

Two of the three patients in whom cytogenetic analysis had failed, showed positive BCR-ABL hybridization signals whereas one Ph -ve patient was negative for BCR-ABL hybrid on interphase nuclei, using FISH technique.

Discussion

Cytogenetic analysis plays a very important role in the diagnosis of CML and as a prognostic indicator for monitoring therapy in these patients. This is attributed to the Ph chromosome, cytogenetic abnormality which is consistently associated with CML. The success of cytogenetic analysis depends on the number of metaphases that can be examined and on the proliferative rate of leukemic cells which may vary from case to case. (Hagemeijer, Adriansen & Bartram. 1986; Secker-walker. 1988) Poorly spread or contracted metaphase chromosomes are difficult to analyze using conventional chromosomal analysis for precise identification of complex chromosomal rearrangement.

FISH in the recent years has developed as a powerful technique for detection of numerical and structural chromosomal abnormalities and has been used to elucidate genetic changes in cancers, prenatal and neonatal detection of genetic diseases. (Human Genome News. 1995) This is a relatively new technique which allows detection of Ph chromosome by use of chromosome specific DNA probes. Unlike standard G-banding technique that requires well-spread metaphases wtih good banding patterns for chromosome analysis, FISH can be used to detect chromosomal abnormalities in poorly-spread metaphases and in interphase cells.

FISH has the unique advantage of being suitably combined with morphologic analysis to improve the accuracy of results. (Anastasi, Vardiman & Rudinsky 1991, Tefferi, Schad & Pruthi 1995) Thus, providing combination of karyotypic and other informations, it is more sensitive, providing interpretable information with the use of interphase cells increasing the chances of detection of chromosomal abnormalities among cells with a low proliferative rate.

The FISH procedure used by the earlier studies were very cumbersome, requiring a number of stringent washing steps, labelling the probes and use of appropriate primary and secondary antibodies for amplification of the fluorescent signals. With the availability of commercial probes and hybridization chambers these steps have been reduced and the procedure has been simplified. Therefore it was necessary to validate the usefulness of these new probe sets in order to determine their accuracy.

At times it is not possible to get well spread metaphases for chromosomal analysis from bone marrow aspirates and from cancer tissues. We have performed an independent evaluation for detection of BCR-ABL hybrid in CML patients using FISH on metaphase spreads and interphase nuclei. The FISH assay confirmed the cytogenetic findings on interphase nuclei and in metaphase spreads in most of the cases analyzed. In specific cases of inadequate number of chromosome spreads we were able to demonstrate/exclude BCR-ABL hybrid using FISH analysis.

One patient in this study was Ph -ve using conventional chromosomal analysis but was BCR-ABL positive using FISH analysis. This shows that cytogenetic analysis has limitations of resolution after certain level and the use of FISH technique as an excellent tool for detection of masked Ph chromosome resulting from submicroscopic chromosomal translocation. A small percentage (5-10%) of patients with a presumptive diagnosis of CML, lack a Ph chromosome and are classified as Ph -ve CML,? (Vander Plas, Grosveld & Hagemeijer. 1991) the majority of these having a normal karyotype. These patients may have a masked Ph chromosome or may be lacking the BCR-ABL rearrangement. Occasional findings of such cases have also been reported. (Kurzrock, Kantarjian & Shtalrid. 1990; Vander Plas et al 1991) The other possibility includes that after treatment patients may revert to normal karyotype after intial presence of Ph chromosome. Chemotherapy induced complete cytogenetic remission with hematologic remission has been reported. (Wodzinsky, Potter & Lawrence. 1989) The utility of FISH studies in detecting the minimal residual disease in such patients has been emphasized. (Bose, Choudary, Saxena & Kucheria. 1997) When the results of conventional chromosomal studies are normal, FISH could be used to identify patients with Ph chromosome resulting from submicroscopic translocation. (Gordon, Dewald & Schad. 1993)

From our study it is clear that FISH provides a reliable enhancement to conventional cytogenetics and an alternative to other molecular techniques. Cytogenetic analysis was unsuccessful in three patients but the same cell suspension could be used to analyze BCR-ABL rearrangement in the interphase nuclei using FISH analysis. This shows the advantage of interphase FISH over conventional cytogenetics in determing the status of BCR-ABL rearrangement in all the cell population, not just those which happen to be in metaphase through culture.

Other molecular methods have been employed to detect residual leukemia cells in CML patients. The Ph chromosome and the rearranged BCR-ABL gene can also be detected by Southern blot analysis and by Polymerase Chain Reaction (PCR), however the sensitivity varies. For example, 1-4% malignant cells in the cell population can be assessed by cytogenetics or Southern blot analysis while one malignant cell in 105 normal cells (Lee, Lee & Kantarjain 1988; Kawaski, Clark & Coyne. 1988) can be identified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Molecular methods are also useful in detecting an early relapse after bone marrow transplantation. (Lion, Henn & Gaiger 1993) False positive PCR results can be derived from contaminating traces of DNA which requires strict adherence to precaution. Contamination is a major disadvantage during amplification of a translocation or a breakpoint using similar set of probes and primers. Recent studies by Thijsen et al (1997) have shown detection of BCR-ABL hybrid using FISH method to be more reliable than the PCR method.

The present study concludes that interphase cytogenetics using FISH can add to the diagnostic power of routine cytogenetic analysis. The overall goal was to employ FISH as an adjunct procedure, then a directed analysis, for optimizing its application

and validating the results. The use of FISH on interphase nuclei and poorly spread metaphases overcomes the difficulties of conventional G-banding of metaphase spreads. However, it cannot replace the gold standards of conventional chromosomal analysis as it cannot exclude other chromosomal aberrations for which the specific probe is not used. FISH analysis is of prime importance for detection of BCR-ABL hybrid resulting from submicroscopic translocation in Ph -ve cases. Molecular cytogenetic technique of FISH is an efficient tool in detection of minimal residual disease, and it is of immense importance in diagnosis and assessement of response to therapy.

Acknowledgments : Authors are grateful to All India Institute of Medical Sciences and the Indo-French Center for Promotion of Advanced Research for providing grant for procuring equipment and software for Fluorescence In Situ Hybridization analysis.

References

Anastasi, J., Vardiman J.W. and Rudinsky R., (1991) Direct correlation of cytogenetic findings with cell morphology using in situ hybridization : An analysis of suspicious cells in bone marrow specimens of two patients completing therapy for acute lymphoblastic leukemia. Blood 77 : 2456

Ben Neriah, Y., Daley G.Q. and Mes-Masson, A., (1986) The chronic myelogenous leukemia specific p210 protein is the product of the bcr/abl hybrid gene. Science 233 : 212

Bose, S., Choudary, V.P., Saxena, R., Kucheria, K. (1997) Lymphoid blast crisis during complete cytogenetic remission following IFN-alpha and Hydroxyurea therapy. Acta Haematologica 98 : 155

Champlin, R.E and Glode, D.W. (1985) Chronic myelogenous leukemia ? recent advances. Blood 65 : 1039

Clark, SS., Mc Laughlin, J. and Crist, W.M., (1987) Unique forms of the abl tyrosine kinase distinguish Ph positive CML from positive ALL Science 235 : 85

Gordon, W., Dewald, C.R. and Schad, E.R., (1993) The application of Fluorescent In Situ Hybridization to detect Mbcr/abl fusion in variant Ph chromosome in CML and ALL. Cancer Genetics and Cytogenetics 71:7

Hagemeijer, A., Adriansen, H.J. and Bartram, C.R. : New possibilities for cytogenetic analysis of leukemic cells Hagenbeek, A. and Lowenberg, B. (ed) In : Minimal residual disease in acute leukemia. Dordrecht, The Netherlands, Nijhoft : 1, (1986)

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Kloetzer, W., Kurarock, R. and Smith., (1985) The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity. Virology 140 : 230

Konopka, J.B., Watanabe, S.M. and Singer, J.W., (1985) Cell lines and clinical isolates derived from Ph positive chronic myelogenous leukemia patients express abl proteins with common structural alterations. Proceedings of National Academy of Sciences; USA 82 : 1810

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V Jobanputra, TA Sivakumaran, K Kucheria Department of Anatomy, All India Institute of Medical Sciences New Delhi-110029

For Reprints, request Kucheria, K.



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