| Abstract |
Lung cancer continues to have by far the highest cancer mortality in the
UK. Beyond stage of disease and the patient's Performance Status there are
no other robust clinical or molecular markers of prognosis available today.
One major reason for the high mortality rate of this disease is the
significant proportion of patients who present with advanced incurable
stage disease. A second significant problem in the management of patients
with lung cancer is chemotherapy resistance. In patients with NSCLC (80%
of cases) initial response rates to cisplatin-based chemotherapy are
modest at best. In the case of SCLC (20% of cases) the initial response
rates to cisplatin-based chemotherapy are high. However relapse, often
with chemotherapy resistant disease, is all too common.
There is a significant body of evidence that demonstrates the role of the
loss of mismatch repair activity in the mechanism of chemotherapy
resistance. Studies to date have demonstrated that the loss of mismatch
repair protein expression is a consequence of 2 main mechanisms: gene
mutation and the epigenetic phenomenon of methylation of the MLH1 (a major
mismatch repair protein) gene promoter region. Studies have shown that the
loss of MLH1 expression is associated with acquired resistance in ovarian
and breast cancers. Allelic imbalance (loss of heterozygosity) of
chromosome 3p is common in lung cancer samples and the MLH1 gene locus
resides here. This loss of heterozygosity (LOH) has been demonstrated to
be a poor prognostic indicator in patients with primary adenocarcinoma of
the lung. In this translational research project the role of mismatch
repair (MMR) and LOH in patients with lung cancer has been further
investigated. In the first study using 2 separate cohorts of patients the
potential role of mismatch repair proteins as a marker of prognosis in
patients with NSCLC was investigated.
The first cohort of patients had received either cisplatin based or non-
cisplatin based chemotherapy for predominantly advanced (stage IIIb/IV)
disease at Stobhill Hospital in Glasgow. Pre-treatment paraffin-embedded
bronchoscopic samples were collected retrospectively. The aims of this
study were to examine the level of expression of the MLH1, MSH2, p53 and
Ki67 proteins in these small samples and assess whether these results
correlated with any clinicopathological variables or with prognosis. In
addition differences in overall survival between the cisplatin and the non-
cisplatin chemotherapy treated patients were assessed relative to the
level of protein expression. Despite the small size of the biopsies good
inter-observer kappa scores were demonstrated between 2 independent
scorers for each protein immunohistochemistry score (IHC) (MLH1 -0.6062,
MSH2 -0.4313, p53 -0.591). Although this demonstrates that historic
bronchoscopy samples can be used for such studies, the number of markers
studied was limited due to small sample size and in this study there was
insufficient tissue to assess expression levels of Ki67. No significant
correlations were demonstrated between IHC score and overall survival for
any of the proteins studied, nor was there any difference between the
different chemotherapy regimens. Similarly there was no correlation
between IHC score of the studied proteins and any clinicopathological
variables.
The second cohort of patients all had resection of their primary NSCLC
tumour at Aberdeen Royal Infirmary. Of these 50 patients, 10 had received
pre-operative cisplatin-based chemotherapy. Fresh frozen surgical samples
collected at the time of surgery were analysed retrospectively. The aims
of this study were to examine the level of expression of the MLH1, MSH2,
p53 and Ki67 proteins in these samples and assess whether these results
correlated with any clinicopathological variables or prognosis. Whether
any difference existed in these variables between the patients receiving
pre-operative chemotherapy and those that did not was also investigated.
This study failed to show any significant correlation between the level of
protein expression and overall survival or any other clinicopathological
variable. Further there was no difference in the level of protein
expression between those patients who had received pre-operative
chemotherapy and those who had not. However given that only a small number
of patients had received pre-operative chemotherapy, further large studies
would be required to validate these results.
In the second study we investigated the status of CpG island methylation
(using methylation specific PCR) and its role as a marker of prognosis in
patients with NSCLC. The samples were the same surgical samples as
described above as well as normal adjacent lung tissue. The markers
studied were MLH1, p16, DAPK, TIMP 3, HIC 1, MINT 25, MINT 31 and RASSF1A.
In this study 30 (60%) of samples exhibited methylation of at least one
promoter site with 19 (38%) at 1 site, 5 (10%) at 2 sites, 2 (4%) at 3
sites and 4 (8%) at 4 sites. Twenty (40%) of the tumour samples exhibited
no methylation at any promoter sites. Methylation rates in normal adjacent
lung tissue were low. There was no significant correlation between the
number of methylated sites and either overall survival or any other
studied clinicopathological variable. The investigation of methylation at
individual sites demonstrated an association between HIC 1 methylation and
stage of disease (p = 0.020) and methylation of MINT 31 was associated
with a better overall survival (P = 0.030). This remained the case when
analysis was performed excluding those patients who had received pre-
operative chemotherapy. This is the first report of MINT 31 methylation
being studied in NSCLC and therefore further independent studies would be
required to validate these results and confirm that the associations had
not occurred by chance due to multiple testing. No significant differences
in any of the studied variables were demonstrated when comparing patients
who had received pre-operative chemotherapy with those who had not.
A third study was performed to validate the findings of previous studies
that loss of heterozygosity (LOH) of chromosome 3p is a common occurrence
in patients with NSCLC (Mitsudomi et al., 1996). This study also aimed to
investigate any correlations between 3p LOH (D3S1289, D3S1300, D3S1304) or
hMLH1 promoter methylation and level of MLH1 expression. In addition this
study attempted to correlate the presence of any molecular changes found
in the serum DNA taken preoperatively from patients with those of their
primary tumour samples. For this study prospective collection of surgical
tumour and normal adjacent lung tissue samples as well as a pre-operative
whole blood sample was collected from patients undergoing resection of
their primary disease at the Western Infirmary in Glasgow. Numbers in this
study were small thus making any attempt at statistical analysis
inappropriate. Observations demonstrated that 3p LOH was common in the
primary tumour with 4/8 samples demonstrating LOH at D3S1289, 5/6 at
D3S1300 and 5/9 at D3S1304. Corresponding changes were demonstrated in the
preoperative serum samples in 2 of the 4 patients at D3S1289, 3/5 at
D3S1300 and 2/5 at D3S1304. Loss of Heterozygosity at chromosome 3p did
not appear to affect the level of MLH1 expression and hMLH1 methylation
was not demonstrated in any of the studied tumour samples.
A final study, in collaboration with Dr J Plumb, was performed to
investigate the role of the mismatch repair proteins in the chemotherapy
sensitivity of a panel of small cell lung cancer cell lines. In this study
it was demonstrated that there was a high correlation between cisplatin
sensitivity and the mismatch repair proteins MLH1 (r2 = 0.83) and MSH2 (r2
= 0.87) but not PMS2 (r2 = 0.22). Two of the cell lines originated from
metastatic biopsies from the same patient, one pre-chemotherapy treatment
(LS274) and one post-chemotherapy (LS310). It was shown that LS310 is 2.3
times more resistant to cisplatin and shows a 50% reduction in MLH1
expression when compared to LS274 (p < 0.001). It was demonstrated that
the hMLH1 promoter region of LS310 exhibited methylation whereas the LS274
promoter region did not. Neither of these lines exhibited methylation of
the p16, MINT 25 or DAPK loci suggesting that de novo methylation was not
responsible for the methylation specific PCR results. Further work
demonstrated that treatment of the LS310 cell line with the demethylating
agent decitabine increased its cisplatin sensitivity as well as increasing
the level of MLH1 expression of the cell line. No such changes were
demonstrated in the LS274 cell line after treatment with decitabine.
In summary, this research project was limited by the availability of
samples. However it has demonstrated that collaborative multidisciplinary
prospective planned translational research can be done and emphasises the
need for a translational component to be an integral part of future lung
cancer studies.
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