Conclusions

1

Human Leukocytes Antigen (HLA) Association with
Brain Astrocytic Tumors Detected by Genomic DNA
Typing
Objectives To determine the prevalence and
magnitude of HLA class I and II alleles in
patients with brain astrocytoma by DNA genomic
typing. HLA-A, B, and -DRB genotypes and
haplotype combinations frequency were also
examined for the same patients compared with
matched controls.
University of Sydney Faculty of
Medicine Childrens Hospital at
Westmead Molecular Pathology Department
Ammira Al-Shabeeb
Hypothesis Brain Astrocytic tumours could be
caused via multiple factors including continuous
exposure to a chemical and radical agents through
the environmental pollution as well as the
genetic factors. On the molecular level there are
certain genetic mutations occurs. we hypothesized
that the loss of HLA single alleles or
combinations could be a predisposing factor for
brain astrocytoma development. Brain astrocytoma
is a major disease, so we designed this study
that demonstrate the association between
HLA-class I II and brain astrocytic tumorus.
Background The Major Histocompatibility complex
refer to Human Leukocyte Antigen (HLA). The loss
of HLA antigens by neoplastic cells is considered
important for tumor growth and metastasis. HLA
system is kind of genetic marker of human being,
and the most complicated human genetic
polymorphic system with hereditary of haplotype
inheritance and allele polymorphism and linkage
disequilibrium. The alleles of HLA system
control a variety of immune functions and
influence the susceptibility to more than 40
disease. Positive correlations between HLA and
cancers have been reported. However, this kind of
correlation has not been studied in patients with
brain astrocytoma in Iraq (Figure 1- A, B, C, D).
Corresponding Author ammira_at_centenary.org.au
ABSTRACT This study investigated the association
between HLA-A, B, DR alleles, and astrocytoma in
Iraqi patients. A total of 30 patients with WHO
grade I, II, and IV astrocytoma were studied (24
males and 6 females). Blood samples were used for
HLA genotyping by PCR-SSP . The allelic
frequencies were calculated and compared with
controls. There are certain HLA alleles that
showed significant association with brain
astrocytoma. The incidence of HLA-A risk group of
alleles was significantly decreased in patients
group by 6 folds risk, thereby assigning a
protective role. Consistent association between
HLA-B alleles and brain astrocytoma, thereby
conferring disease susceptibility. Regarding to
HLA-DRB locus, there was a significant decrease
in the frequencies of risk group alleles in
patients and controls. The importance of HLA-A,
B, DRB1 risk group of alleles was more striking
in certain HLA combinations. Individuals with
HLA-A03012, which were in linkage disequilibrium
with HLA-B3804, had an increased risk of brain
astrocytoma ( CCRV 15.34). These data suggested
that there were significant association between
certain HLA single alleles with brain astrocytoma
and combinations were associated with a
significant risk for astrocytoma. Thus, they may
predispose to developing this malignant disease.
These associations may serve as indicative for
the involvement of the immune system of the host
anti tumour surveillance, recognitions, and
destruction of human tumour cells.
Methodology  T Eighty-three brain tumor biopsies
were collected and used in this study. Thirty
were diagnosed as brain astrocytoma as confirmed
by the histopathological examination. HE slides
were prepared for the paraffin embedded blocks
for histological typing and grading. T Specimens
from thirty patients with astrocytoma were
selected to be investigated in more details. All
showed astrocytic brain tumors of different
grades and stages. Sex and age matched
individuals were also included as a healthy
controls. T Genomic DNA was isolated from
fresh blood samples. T PCR-SSP Analysis was
used to measure the association between HLA-A,
B, DRB alleles and the incidence of
astrocytoma. T Standard Statistical analysis
methods were adopted throughout this study.
RESULTS
RESULTS
Figure 1- A
B
Table 1 Allelic association at class I HLA-A
locus. The Relative Risk (RR) of 0.842 for HLA-A
alleles and astrocytoma act as a cut-off value
which represent the lower limit of 99 confidence
interval (CI).
C
D
RESULTS
Table 2 Allelic association at class I HLA-B
locus. The cut off value of the odds ratio was
0.14, which represent the lower limit of the 99
confidence interval (CI).There was high relative
risk values for some lost alleles that might make
them more associated with disease occurrence than
the rest.
Figure 2 PCR-SSP HLA-A, -B, -DRB genotyping of
astrocytoma patient (ID17). Genomic DNA was
extracted from blood. Electrophoresis was carried
out in 2 agarose at 8V / cm for 30 minutes and
stained with ethidium bromide. Lanes (1-24)
represent class I HLA-A alleles (25-72)
represent class I HLAB alleles (73-95) represent
class II HLA-DRB1, 3, 4, and 5 alleles,
respectively (96) represents the negative
control. Internal positive control a fragment of
human growth hormone 1069 bp. This genotyping was
obtained by using primers that detect the
following alleles as they were present in the
numbered wells, respectively. Patient (ID17)
HLA-A, -B, -DRB Genotyping A1, 36 A2 A3 A11
A23(9) A24(9) A10. A25(10) A26(10),
43A34(10) A6602, 6603A10.2A36A43A68(28A69(2
8)A29,31,32,33,74A30(19) A31(19) A 32(19) A
33(19) A 74(19) A 80 B 7,703 B8 B13
B64(14), 65(14) B15 without B75 B15without
B63 B63(15) B70, 71(70), 72(70), 15 B62, 63,
75, 77 B75, 35,15 B76 B18 B27, 2708
B7,8,14,35, 39, 42, 48, 67, 78, 81 B8, 35, 51,
53, 78 B18, 35, 78 B73 B38(16) B38(16),
39(16), 67 B60 (40), 48, 81 B60 (40), 61 (40),
47 B8, 41, 42 B44 (12) B45 (12) B46 B47
B48 B49 (21) B49 (21), 50 (21), 51(5) B72
(70), 50 (21), 40, 41, 45 B51 (5), 5102/03, 52
(5) B51(5 ,5102/03, 52(5)B52 B52.1 B8, 38,
51(5), 52(5), 53, 58 B7, 27, 42, 46, 54, 55,
56(22) B54(22) B7,42,55,56(22),67 B54(22),
55(22) ,39 ,41 ,59 B56(22) B57(17)B13,59 B73
B62(15), 70, 78 Bw4 Bw6 DR1 DR103 DR15
DR16 DR17 DR18 DR4 DR7 DR8 DR9 DR10
Dr11 DR12 DE13.1 DR13.3 DR13.4 14.1 DR14.2.
Table 5 Corrected Cumulative Risk Value (CCRV)
of the risk group of alleles. Further analysis
was carried out for the risk group of alleles by
using CCRV as a parameter to express the risk in
patients when two like ( HLA-A HLA-B, HLA-A
HLA-DRB, HLA-B HLA-DRB) or three of the loci
are combined like( HLA-A, B, DRB). The CCRV was
calculated as the sum of the two or three
relative risk (RR) values of the alleles after
deducing the mean of the risk value of each of
the HLA loci from the respective allelic risk
value as a correction factor. The highlighted
areas represent the highest CCRV which observed
in all haplotypes combinations.
Table 3 Allelic association at class II
HLA-DRB1 locus. The cut off value was 0.643,
which represent the lower limit of the 99
confidence interval. The neutral group included
all odds group included the alleles with odds
ratio that represented 6.5 folds of cut-off
value. The risk group allele represented 13.5
folds of the cut-off value. ratio values from
zero to 0.64.. The predisposing allele.
Figure 3 Representation of Corrected Cumulative
Risk Values (CCRV), A HLA-A and HLA-B
cumulative combinations, the cut-off value 5.7.
accordingly, the frequency distribution was
divided into three major risk combination groups,
risk combination group (I) having a RR below the
cut off value (24 combinations, 40.7), risk
combination group (II) having RR above the
cut-off value (24 combinations, 40.7), and the
high-risk combination group (11 combinations,
18.6). B HLA-A HLA-DRB1 cumulative
combinations, the cut-off value 8.3. risk
combination group (I) (56 combinations, 25.5),
risk combination group (II)( 140 combinations,
63.6), and high-risk combination group (24
combinations, 10.9). C HLA-B HLA-DRB
cumulative combinations, the cut-off 6.3, the
risk group combination (I)(44 combinations,
53.7), risk group combinations (II) (32
combinations, 39), high-risk combination group
(6 combinations, 7.3). D HLA-A, HLA-B HLA-DRB
cumulative combinations, the cut-off 10.3, the
risk combination group (I)(252 combinations,
32.7), risk combination group (II)(446
combinations, 57.9), the high-risk combination
group (72 combination, 9.4).

• Conclusions

Table 4 Corrected Cumulative Risk Value (CCRV)
for HLA class I and II risk group alleles
combinations. A the number of haplotypes in this
combination 59, mean (6.86), Cut off value
(5.74). B the number of haplotype combinations
220, mean (8.33), cut off value (8.09). C the
number of haplotype combinations 82, mean (7.28),
cut off value (6.30). D the number of haplotype
combinations 770, mean (10.6), cut off value
(10.3).
O- Loss of single HLA-A,-B, and DRB1 alleles and
their combinations and estimated haplotypes
showed significant association with
astrocytoma. O- HLA-B124415 and B4420 alleles
were significantly more common in patients with
astrocytoma than in health controls (highest OR
4.24). They could be considered as risk markers.
Thus, individuals carrying these alleles might be
susceptible to brain astrocytoma. O- A
considerable port of the astrocytoma tested
showed loss of HLA-A and B alleles, which may
enable them to escape from the immunosurveillance
of cytotoxic T-cells.
Table 6 Expected and Observed probabilities of
HLA A, B , DR loci (A) and HLA-A, B, DRB1
risk group combinations (B). The observed
probability higher than expected for HLA-A, B,
DRB1 loci combinations. This indicates that these
alleles might be decisive factors in disease
initiation.
ACKNOWLEGMENT Dr. Hassan M. Naif Molecular
Pathology Head Childrens Hospital _at_ Westmead
Sydney Cancer Conference, 24 26 July
2008 University of Sydney
Special Thanks to Dr. Silvia Ling, Cancer Drug
Resistance Group, Centenary Institute of Cancer
Medicine and Cell Biology.