الفهرس 
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Abstract
Chronic lymphocytic leukemia is often associated with an increased frequency and
severity of infections mediating the immune disturbance in CLL patients. This suggested
that common infections may play a role in CLL etiology. The present work was conducted
to study the total expression of HLA-G and TLR-9 in CLL patients, evaluating their
relationships with other well established clinical and possible laboratory prognostic
markers.
This study was conducted prospectively for 40 CLL patients (26 male and 14
female patients) selected from the Hematology Department of the medical research
institute and Alexandria Main Hospital during the period from Feb.1st to April. 30th, 2017.
In addition to 20 healthy subjects matched by age and sex.
Patients were selected after diagnosis of CLL who scored 4 or 5 by Matuets’ score
immunophenotyping, with no other hematological disorder and no previous history of
chemotherapeutic drugs, excluding those with hepatitis B or C virus, HIV, bilharziasis, any
autoimmune diseases or other causes of hepatosplenomegaly.
They were subjected to full medical examination, routine laboratory investigations
for hematological and biochemical markers, imaging, staging, CLL prognostic index. Fresh
samples were taken for the detection of surface HLA-G expression and intracellular
expression of TLR-9 by flow cytometry. The control group investigated for complete
blood picture, routine investigations and detection of HLA-G and TLR-9 at the same time.
HLA-G expression on CD5+B cells was evaluated by dual-color
immunofluorescence staining using phycoerythrobilin (PE)–conjugated antihuman HLAG–
specific monoclonal antibody MEM/G9 (BioLegend PE anti-human HLA-G, San Diego
-USA). The intracellular expression of TLR-9 on CD5+ CD19+ CLL cells was performed
after permeabilization using with Fix/Perm Kit (abcam, AntipTLR-9 antibody [5G5]
(FITC) ab58864, United Kingdom).
This study showed that the M: F ratio for CLL was 1.86: 1. Lymphadenopathy was
reported among 87.5%, splenomegaly among 65% and hepatomegaly among 22.5%. CLL
patients showed significantly lower hemoglobin concentration and platelets count, and
higher TLC, ALC, β2-microglobulin, LDH and C-reactive protein when compared to the
control (p<0.05).
Surface expression of HLA-G was variable, ranging from 0 to 21% in CLL patients
and from 0 to 0.08% in the control with significantly higher median expression among
CLL patients (7.5% vs. 0.020%).
Intracellular TLR-9 expression showed lower percentages among CLL patients,
ranging from 0 to 7% while in the control it ranged from 0 to 11% with significantly lower
median expression among CLL patients (2.70% vs. 4.75%).
Both markers (HLA-G and TLR-9) were neither showing significant variable
expression as regard to sex of CLL patients nor to the studied clinical findings, apart of
HLA-G median expression in patients with hepatomegaly.
Summary
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Clinical staging of the studied CLL patients showed higher Rai stage II and III
(35% and 32.5% respectively) and higher Binet stage C (42.5%). Positive expression of
ZAP-70 was reported among 45% of CLL patients. It was higher among late Rai stages (III
and IV; 66.7%) and advanced Binet stage (C; 66.7%).
The median HLA-G expression was statistically significantly increasing with
increasing stage of CLL (p<0.05). For Rai staging; it was 1.5% in stage I, increasing to
5.5% in stage II, 10.0% in stage III and reaching 16.0% in stage IV. For Binet staging; it
was 0.045% in stage A, increasing to 6.0% in stage B and reaching 10.0% in stage C.
On the other side, the median TLR-9 expression was statistically significantly
decreasing with the increasing stage of CLL (p<0.05). For Rai staging; it was 3.7% in stage
I, decreasing to 3.65% in stage II, 1.6% in stage III and reaching 0.2% in stage IV. For
Binet staging; it was 3.6% in stage A, decreasing to 3.3% in stage B and reaching 1.5% in
stage C.
The median expression of HLA-G was statistically significantly higher among
ZAP-70 positive CLL patients while the median intracellular expression of TLR-9 was
statistically significantly lower among ZAP-70 positive CLL patients.
As regard to the CLL clinical risk groups, the median expression of HLA-G was
1.0% in low risk group, reaching 9.0% in intermediate risk group and 15.5% in high risk
group (p<0.05). While the median TLR-9 was 4.5% in low risk group, decreasing to 2.0%
in intermediate risk group and 0.16% in high risk group (p<0.05).
The ROC curve showed significant area under the curve for HLA-G (AUC= 0.841,
p=0.001) and for TLR-9 (AUC= 0.287, p=0.022). This denotes that both markers are
sensitive indicator for positive ZAP-70 state in CLL patients.
Correlation of the HLA-G expression to other markers in CLL patients was
significantly positive to TLC (r=0.507), ALC (r=0.483), β2-microglobulin (r=0.457), LDH
(r=0.504), ZAP-70 expression (r=0.752) and the CLL prognostic index (r=0.591). It was
significantly negative to hemoglobin concentration (r = - 0.472), platelets count (r = -
0.390) and TLR-9 expression (r = - 0.712).
Correlation of the TLR-9 expression to other markers in CLL patients was
significantly positive to hemoglobin concentration (r = 0.431) and platelets count (r =
0.312). It was significantly negative to TLC (r= - 0.442), ALC (r= - 0.435), β2-
microglobulin (r= - 0.315), LDH (r= - 0.366), ZAP-70 expression (r= - 0.584) and CLL
prognostic index (r= - 0.540).
The present study provides evidence to indicate that this distinct pattern of HLA-G
and TLR-9 functional activity in CLL cells might prove relevant for elucidating the
immune mechanisms underlying the natural history of CLL and define subgroups of
patients who might benefit from treatment with specific HLA-G and TLR-9 legends.
This study concluded that expression of HLA-G and TLR-9 could have a prognostic
value in CLL patients. The higher HLA-G expression, the lower TLR-9 expression, the
worst the outcome. The use of HLA-G and/or TLR-9 as markers for new targeted therapy
in CLL could be valuable.