Oncology study i did in my third year (2014). the study was basically about monitoring Chronic Myeloid leukemia (CML) using Real-Time PCR techniques to check how patients from Universitas Hospital responded to the treatment of Gleevec drug.

1. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 1
CLINICAL DIAGNOSIS AND MONITORING OF CHRONIC MYELOID LEUKEMIA BY
REAL TIME POLYMERASE CHAIN REACTION AND FISH
Teboho Mooko, 2011018955
CML is a disease that is caused by a translocation between chromosome 9 and 22. The
clinical diagnosis requires the detection of t(9,22) in the blood or bone marrow samples.
t(9,22) causes BCR-ABL fusion gene which encodes an abnormal protein kinase which send
abnormal signals to the nucleus for the abnormal proliferation of cells in the myeloid
pathway. Immatinib (Gleevec) is mainly used to treat this disease and its mechanism is
based on the inhibition of abnormal Protein kinase signals. In this study, blood samples from
four patients were analyzed to determine the concentrations of BCR-ABL gene. Diagnosis
was done using RT-PCR technique whereby BCR-ABL gene was amplified and measured,
after the successful extraction of RNA from leukocytes. Quantification results from RT-PCR
helped to conclude that Samples from Patient A and C tested positive, but contained low
BCR-ABL gene copies, which was a sign that these patients are responding well to the
treatment although the progression still need to be monitored after every six month by
using FISH technique, which uses probes or fluorescence dye to mark the target DNA
sequence which reflects light when visualized on a light microscope. Sample from Patient B
tested negative for the presence of BCR-ABL gene. Sample from Patient D tested positive for
the presence of BCR-ABL gene which appeared in high concentrations.
Key terms: Chronicle myeloid leukemia (CML), Real-Time Polymerase Chain Reaction (RT-PCR), BCR-
ABL (Fusion gene), Translocation between chromosome 9 and 22 (t(9;22)), Polymerase Chain
Reaction(PCR), fluorescent In Situ Hybridization (FISH), Immatinib (Protein Kinase Inhibitor) ,
Philadelphia chromosome (Ph),
Introduction
(Swayers, 1999) Wrote that, Chronic
Myeloid Leukemia (CML) is a malignant
clonal disorder that occurs over a long
period of time and affects the myeloid
pathway that forms Red blood cells and
platelets in the peripheral blood. He
further wrote that, CML presents in early
fifties, affect children and all age groups
and show the following medical symptom
in individuals: splenomegaly (the
enlargement of the spleen) which is
common in most patients; anorexia;

2. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 2
fatigue; and weight loss. He also stated
that CML can show different phases which
includes blast crisis that often come
before an accelerated phase in which
increasing doses of hydroxyurea
(antineoplastic drug) are required to
lower the neutrophil count; chronic phase
which includes the maturation of CML
cells and the accelerated phase. (S
Brandford, et al. 1999) added that
Bulsanfan drug is also used to treat
patients in a chronic phase, but is limited
to patients who are resistant or intolerant
to hydroxyurea.
(Schoch C, 2002) Indicated that, CML is
characterized by BCR-ABL rearrangement
that is caused by translocation between
long arm of chromosome 9 and long arm
of chromosome 22, which forms the
Philadelphia (Ph) chromosome which was
first discovered in 1960, described as a
“shortened chromosome 22” and
“t(9;22)” in 1973. He further indicated
that, approximately 95% cases of CML in
patients are proved by the presence of Ph
chromosome and approximatley 5% cases
have translocations involving other
proteins besides chromosome 22 and 9.
He also added that the BCR-ABL gene
encodes a protein called Tyrosine kinase
which plays a crucial role in the
pathogenesis and treatment of CML.
(Palka G, 1997) Mentioned that,
cytogenetic analysis is a commonly used
method in the diagnosis of CML and the
demonstration of the BCR-ABL gene is
important in Diagnosis of CML and also to
exclude the presence of Ph negative
which is linked to severe outcome and bad
response to treatment. He further
mentioned that the presense of Ph
negative chromosome can be detected by
using Flourenscent Insitu Hybridization
(FISH) which is able to detect the BCR-ABL
fusion gene on metaphase or on nuclei.
These makes FISH the best tool for clinical
diagnosis and monitoring of CML and to
monitor the progression of CML.
According to the (university of Utah
Health Care), Real Time PCR is a very
sensitive technique used to detect low
levels of BCR-ABL fusion gene in a test
sample. It works by amplifying the amount
of genetic material (specific sequence of
n). It require the mixture of RNA
template, Primers (Hexamers), nucleotide

3. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 3
bases, and enzyme Reverse transcriptase
which help convert RNA into
complementary DNA, Followed by a series
of Denaturation, annealing and
amplification of targeted sequences of
nucleotides which end up giving out
fluorescent light
According to (Speicher, 2005) FISH is a
technique that uses DNA probes which are
flouroscently labelled to hybridize a
sequence of interest arrested in
metaphase of mitosis. The first step of
Hybridization includes denaturation of
DNA molecule, labelling by probes or
florescent dye and incubation and
microscopic visualization of fluorescent
light.
The AIM of this study is the to extract
BCR-ABL fusion gene from blood samples
of four patients, amplify the fusion gene
using qPCR and quantify the
concentrations of the fusion gene to
identify t(9,22) which will help us in
diagnosing and monitoring CML.
Methods and Material
Prior to analysis, Peripheral Blood samples
used in the study were collected from four
patients with CML at hematology clinic,
Universitas hospital. Ethical issues
concerning Clinical research were
followed and contract of agreement
between patients and my senior lecture
was signed. RNA stabilization was
performed on the day of collection.
Analysis of all samples was performed in
the Haematology undergraduate
laboratory and the study was led by Dr
Marx, Senior researcher in Molecular
Genetics.
Triozol stabilization for RNA was done by
adding 5ml of peripheral blood and 25ml
of working lysis buffer into a 50ml tube to
make up the volume of 30ml.the solution
was mixed and incubated at room
temperature for 10 minutes. The mixture
was centrifuged at 3500rpm-4000rpm for
10 minutes to pellet the white cells. The
supernatant (lysed RBC) were carefully
discarded without disturbing the white

4. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 4
pallet that was at the bottom of
centrifuge tube. 5ml of working lysis
buffer was added to make a 50 ml volume
and the pellet was re-suspended using a
sterile transfer pipette. The mixture was
incubated at room temperature for 5-10
minutes and centrifugation was repeated.
The supernatant was discarded leaving
the white cell pallet. 800ul of Trizol
solution was added using a sterile transfer
pipette and mixed by rinsing in and out.
The solution was incubated at room
temperature for at least 40 minutes.
RNA was extracted from a Trizol
Homogenate whereby 15ul of Proteinase
K was added to Thaw homogenate and
incubated at 65˚C for 20 minutes. 350ul of
chloroform was added to 1.6ml thawed
trizol homogenate. Solution was vortexed
for 10s and placed on ice for 3 min;
followed by centrifugation at 14000 rpm
for 15 min. 2×500ul of the supernatant
was transferred to a 2ml of Rnase and
DNase free tube. Iso propanol was added
to the supernatant and incubated on ice
for 30 min, followed by centrifugation at
12000rpm for 10 min. The supernatant
was discarded and RNA pallet was washed
with 1ml of 75% ethanol by vortexing for
10s and centrifuged at 10 000 rpm for
10min.ethanol traces were removed by
inverting the tube on a paper towel. 40-
50ul of DEPC was added and dissolved in
Rnase and Dnase fee water for 15min at
55˚C. RNA concentration were
determined with Qubit fluorometer.
RNA Concentrations were determined by
mixing 1ul of extracted RNA with a buffer
and flourophore. Mixture was incubated
at room temperature for 2min.Quanti-iT
kit for RNA assay was used in determining
RNA concentrations in ug/ml. all
Procedures required to run Qubit
flourometer were followed correctly and
appropriate Quanti-IT working solutions
and probe were added. The readings
obtained in ug/ml were converted to
ng/ul by multiplying with the dilution
factor (0.2for sample A,B, and D and 0.3
for Sample C) and the extra working
solution added in ul devided by 1000.
After concentrations were recoded, RNA
aliquots were made in a 0.6ml micro-
centrifuge tube containing 2000ng/ul-
4000ng/ul of RNA. tube were labeled with
date and amount of RNA in ul.

5. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 5
RNA Visualization The concentration of
200ng/ul of RNA was dispensed into a
labelled 0.6ml micro-centrifuge tube.5ul
of loading buffer was added and loaded
onto a 1% agarose gel. The gel was ran
and stained in Ethidium bromide. The gel
was viewed on the gel Geldoc system and
the picture was taken.
cDNA synthesis was done prior to RT-PCR,
total extracted RNA was primed with gene
specific primers for both BCR-ABL and the
control gene (ONLY RANDOM
HEXAMERS). A reverse transcriptase
cocktail was prepared according to the
protocol and added. The reactions were
incubated at 42˚ for 60 minutes after the
time reverse transcription was stopped at
70˚C for 10 minutes. This cDNA was used
as template material during real-time PCR
for absolute quantification of BCR-ABL
expression. The appropriate reagents
were used and used according to the
research proposal.
BCR-ABL quantification using Real Time
PCR was done by extracting 5ul of the
Cdna template. The RT-PCR grid was
completed to clearly indicate individual
reactions which include, 1 cycle ran at
95˚C for 10 minute, 40 cycles at 95˚C for
15s and 40 cycles at 60˚C for 1 minute.
GUS and BCR-ABL were determined with
separate reactions. Sample for each
patient were run in duplicate, thus each
patient sample underwent 4 individual
reactions. RT-PCR cocktail for 1 sample
was mixed with 12.5ul of TaqMan®
universal master mix, 0.5ul of forward and
reverse primer at a final concentration of
0.2uM, 0.25ul probe at a final
concentration of 0.1 uM and 6025ul
SABEX Water. Separate cocktails were
prepared for GUS and BCR-ABL and were
mixed with RT-PCR cocktail by gently
pipetting. 20ul of reaction was Aliquot to
each tube. 5ul of template cDNA was
added to each reaction and all solution
was ejected from the pipette tip. Tubes
were tightly closed and briefly centrifuged
and inserted into the RT-PCR according to
the layout on the grid form. After
completion of the run, analysis was made
and standard curve had R2
> 0.98.

6. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 6
Results
M A B C D
Figure 1 Agarose Gel picture viewed on the
Geldoc system. M is the marker and A-D
represents different sample quantity and
quality of RNA.
Table 1 : RNA concentration readings as
given by the QUBIT FLUOROMETER in ug/ml
Readi
ng
Sam
ple A
Sam
ple B
Sam
ple
C**
Sam
ple D
1 286 181 570 314
2 292 180 570 320
3 289 181 560 300
Avera
ge
289 181 567 311
Table 2 : with the use of 1:5 ratio of dilution, concentrations of patients were calculated by
multiplying the average (as given by the QUBIT FLOUROMETER) of sample A,B and D with 0.2 and
sample C with 0.3 because of an extra 100ul of buffer that was added. 0.2 OR 0.3×5×(AVERAGE IN
ug/ml) = concentration in ng/ul.
Reading Sample A Sample B Sample C** Sample D
1 286 181 570 314
2 292 180 570 320
3 289 181 560 300
Average 289 ng/ul 181 ng/ul 851 ng/ul 311 ng/ul

7. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 7
Table 3: GUS control gene on well A1,B1 AND C1, and BCR-ABL gene on well G,H, and D.
Undetermined CT values means that there is no contamination and no duplication needed.
Reading for each sample done in duplicate.
Sample
Name
Target
Name
Cт Cт Mean Quantity Quantity Mean
A3 A-1 GUS 24.0353241 24.0353241 80380.44531
C3 A-1 BCR-ABL 36.59493256 36.59493256 78.20074463
B3 A-2 GUS 23.8974762 23.8974762 88141.52344
D3 A-2 BCR-ABL 36.7503624 36.7503624 71.18066406
A1 GUS C1 GUS 30.48130798 30.48130798 1000
B1 GUS C2 GUS 27.38071823 27.38071823 10000
C1 GUS C3 GUS 23.59394073 23.59394073 100000
A2 GUS NTC GUS Undetermined
E3 B-1 GUS 22.29449272 22.29449272 257440.5625
G3 B-1 BCR-ABL Undetermined
F3 B-2 GUS 21.97352219 21.97352219 319069.1563
H3 B-2 BCR-ABL Undetermined
A4 C-1 GUS 24.24935341 24.24935341 69662.26563
C4 C-1 BCR-ABL 35.7075119 35.7075119 133.7937317
B4 C-2 GUS 24.24663162 24.24663162 69789.16406
D4 C-2 BCR-ABL 35.87691498 35.87691498 120.7576294
E4 D-1 GUS 24.431036 24.431036 61693.31641
G4 D-1 BCR-ABL 28.03449059 28.03449059 13899.47949
F4 D-2 GUS 24.79836082 24.79836082 48257.99609
H4 D-2 BCR-ABL 28.32896805 28.32896805 11630.71191
Table 4 : shows the percentage of BCR-ABL in patients samples, percentages were obtained by
dividing the target copy number(BCR-ABL) with Reference copy number (GUS) and multiplied by
100. Sample A and C indicates that a patient is in remission (responding well to treatment), sample
B no BCR-ABL detected, sample D: a patient is not responding well to immatinib or diagnosis was
made later, thus cml is present.
SAMPLE REF BCR-ABL %BCR-ABL AVE%BCR-ABL SD
A-1 80380.45 78.20 0.10 0.09 0.01
A-2 88141.52 71.18 0.08
B-1 257440.56 Undetermined Undetermined Undetermined
B-2 319069.16 Undetermined Undetermined
C-1 69662.27 133.79 0.19 0.18 0.01
C-2 69789.16 120.76 0.17
D-1 61693.32 13899.48 22.53 23.32 1.11
D-2 48258.00 11630.71 24.10

8. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 8
ANALYSIS
The mrna was successfully extracted from
the blood and the concentrations were
determined effectively. mrna from four
patient samples was loaded and run on an
aragose gel. Different migrations of
mRNAs are presented on figure 1, with
the smears because PCR was not yet run.
Mrna had a good quality and quantity.
Sample C appears to have high mrna
concentrations. Table 1 shows mrna
concentrations recorded from the Qubit
Flurometer and are expressed in ng/ul.
Table 2 presents the converted average
concentrations of samples from
flurometer in mg/ul. Evidence from table
3 (CT mean) suggest that Samples named,
A-1, C-1, AND D-1 had BCR-ABL gene and
for Sample B-1, BCR-ABL was
undetermined. CT mean values for GUS
and BCR-ABL gene were below 36. Table
4 clarifies that, sample A and C had BCR-
ABL concentrations of approximately
equal to 0, suggesting that Patient A and C
are responding well to treatment, thus
they are on remission. Sample B indicates
that CT mean values were undetermined
for BCR-ABL copy target number, thus the
patient might have undetectable BCR-ABL
translocation that can be detectable by
cytogenetic techniques if present. (Schoch
C, 2002). Sample D designate high CT
mean values, suggesting that the patient
has high levels of BCR-ABL amount, thus
the patient is not responding well to the
treatment (Gleevec) and cells with t(9,22)
are still proliferating due to increased
synthesis of abnormal tyrosine kinase.
Conclusions
To diagnose CML, the extracted mRna
which has sequence of BCR-ABL was
further analyzed with an RT-PCR
technique to determine the percentages
of BCR-ABL copies in each patient’s blood
sample. In reference to the control gene
GUS, the technique helped us determine
the copy number of BCR-ABL gene with
the reaction repeated twice for each
sample. The results were obtained from
RT-PCR 7500 Software v2.0.5 of which the
experimental menu was set as CML
quantitative template, type: standard
curve and for reagents: Taqman reagents.
The run took about 2 hours before the
results were made. Calculations from
these results suggested that the BCR-ABL
percentages for sample A and C were
approximately equal to 0, which was an
indication that patient A and C were
responding very well to treatment, due to
low copies of BCR-ABL gene. For Sample B,
the BCR-ABL gene was undetermined,
suggesting that Patient B has no t(9,22)
and sample can further be used to test for
some translocations that RT-PCR cannot
determine. Sample D had increased
percentages of BCR-ABL gene, suggesting
that Patient D is not responding well to
treatment or not taking the treatment as
prescribed or was diagnosed later after
he/she accumulated t(9,22), thus CML is

9. Chronicle Myeloid Research Vol 1 No 1
©2014 Department of haematology and cell biology,
University Of the Free State, 9301, RSA
October 13, 2014 Chronicle Myeloid Leukaemia Research Page 9
still progressive. PCR is an important tool
in the diagnosis of CML. Fish is important
in Monitoring CML
Reference list:
Palka G, B. B. (1997). Routine Flourescence In
Situ Hybridization analysis for
detection of BCR-ABL rearangement
in Myeloproliferative disorder.
Leukemia Research, 21(6), 3.
Schoch C, B. S. (2002). Comparison of
chromosome banding analysis,
interphase and hypermetaphase-
FISH,qualitative and quantitative PCR
for diagnosis and for follow up in
CML: a studt of 350 cases. Leukemia,
7.
Speicher, M. e. (Ed.). (2005). Scietable by
education. (Nature) Retrieved october
09, 2014, from Scietable website:
http://www.nature.com/scietable/co
ntent/principles-ofFlourenscent-in-
situ-hybridization-35120
Swayers, C. L. (1999, April 29). Medical
Progress of Chronic Myeloid
Leukemia. The new England Journal of
Medicine, 340(17), 11.
university of Utah Health Care. (n.d.).
Retrieved october 09, 2014, from
university of Utah health care library:
http://www.healthcare.utah.edu/heal
thlibrary/related/doc.php?type=34BC
MLD7
S Branford, TP Hughes and Z Rudzki (1999)
Monitoring chronic myeloid leukaemia
therapy by real-time quantitative PCR in
blood is a reliable alternative to bone
marrow cytogenetics. British Journal of
Haematology, 107:587-599.
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