An immunohistochemical and microsatellite analysis of nephroblastomas.
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Date
2008
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Abstract
The aims of this study were: (i) to determine the association between p53, bcl-2, pRb,
p21, cyclin A and p-glycoprotein immunoexpression and prognosis, and (ii) to
determine the frequency of loss of heterozygosity and microsatellite instability at 11 p,
16q and mismatch repair gene loci and their association with prognosis, in
nephroblastomas in South African children.
There were 138 cases (111 of whom received preoperative chemotherapy) in the
immunohistochemical study and, 70 cases (48 with preoperative chemotherapy) in
the microsatellite study. The following monoclonal antibodies were used after heat
induced epitope retrieval; p53, bcl-2, pRb, p21, cyclin A and p-glycoprotein. Six
polymorphic microsatellite markers were selected from the 11p region, 5 from the 16q
region and 6 from the loci of known mismatch repair genes. Automated fluorescent
DNA technology was used in the analysis. The results of the immunohistochemical
and microsatellite studies were correlated with patient age, gender, preoperative
chemotherapy, SlOP histological classification, SlOP histological risk group,
clinicopathological stage, patient outcome and survival using X2
, Fisher's exact test,
Cox regression model and Kaplan-Meier estimates.
The majority of patients presented with advanced disease. Anaplastic tumours and
high-risk histology were associated with high disease stage. Mortality was directly
related to increasing stage and histological risk group. Multivariate analysis showed
that clinicopathological stage was the only factor significantly associated with survival
(p<0.001) (hr=5.6, 95%CI: 2.1-14.9).
High expression of p53 was more frequent in anaplastic tumours suggesting that p53
mutations are common events in this tumour type (p<0.001). Despite the strong
association with tumour histology, there was no association with stage. Although p53
expression was found to be a predictor of survival in the univariate analysis this was
not retained in the multivariate analysis. Tumours treated with preoperative
chemotherapy showed higher bcl-2 immunoreactivity (p=0.027 but lower levels of pRb
(p=0.040) and cyclin A expression (p<0.001). All anaplastic tumours showed high
expression of pRb compared to the other histological types (p=0.003). Expression of
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pRb was significantly associated with survival in the univariate analysis but not in the
multivariate analysis. High cyclin A expression was associated with high risk histology
(p<0.001). Cyclin A expression was found to be a significant predictor of survival in
both the univariate (hr=1.7; 95%CI 1.2-2.4; p=0.002) and multivariate analyses
(hr=1.7; 95%CI1.1-2.7; p=0.032). Although tumours with high risk histology were
more likely to express high levels of p-glycoprotein, this did not reach significance.
LOH at 11 p was seen in 64.7% of 68 informative cases. LOH at 11 p13 was more
frequent than LOH at 11p15. LOH for both 11p13 and 11p15 was found in 39.7% of
all tumours. MSI at 11 p was seen in 22.1 % of informative cases. The majority
showed MSI for one marker only. LOH 16q was seen in 66.7% of 66 informative
cases. MSI at 16q was seen in 16.7% of cases. LOH for 016S496 and 016S520
appear to be related to tumour histology and risk group. The most frequent locus for
LOH was 16q21-22, which is known to harbour important genes, such as, E2F4 and
E-cadherin. LOH for MMR markers was seen in 43.5% of 69 informative cases. MSI
was seen in 11.6% of tumours. In the multivariate analysis there was no significant
correlation between LOH at any of the loci studied and survival. There were no
tumours with high frequency MSI. Low frequency MSI was of no clinicopathological
significance.
The following conclusions are made: (i) p53 mutations determined by high p53
expression is a frequent finding in anaplastic tumours, (ii) Bcl-2 may play a role in the
chemoresistance of nephroblastomas, (iii) Rb gene alterations are not important in
the development of nephroblastoma and anaplasia, (iv) Cyclin A expression is an
independent predictor of survival, (v) p-glycoprotein may be responsible for the
chemoresistance in a proportion of nephroblastomas, (vi) MSI is a rare occurrence in
nephroblastoma and does not play a role in the development of nephroblastoma, (vii)
LOH at 11 p and 16q are frequent findings in nephroblastomas, (viii) LOH for the
specific 16q markers (016S496 and 016S520) may have an important prognostic role
in nephroblastoma.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2008.
Keywords
Immunohistochemistry., Nephroblastoma., Microsatellites (Genetics), Histology., Theses--Anatomical pathology.