| Abstract |
INTRODUCTION
Despite advances in diagnosis and treatment, colorectal cancer (CRC) remains a major public
health problem. It is estimated that one million new cases are diagnosed worldwide every
year. The majority of the cases are not associated with any underlying condition while
recognized risk factors for CRC are male sex, age, bowel polyps and various environmental
factors.
The last decade there has been a marked improvement in the survival of patients with CRC.
This is partly due to the improved screening and early diagnosis methods and also due to
the more efficient surgical and pharmacological treatment options. The guaic-based feacal
occult blood test and sigmoidoscopy / colonoscopy are the preferred screening tools. On the
other hand efficacy of pharmacological treatments is increased as a result of our better
understanding of the molecular mechanisms of cancer that (a) aid in the development of
novel compounds and (b) help identify groups of patients more likely to benefit from
specific treatments.
The optimal treatment for the newly diagnosed patient with CRC is surgical resection of the
primary tumour, whenever this is possible, followed by the administration of systemic
chemotherapy. In the recent years a new class of drugs, the targeted therapies (TT), have
shown to be beneficial in patients with CRC. One of the advantages of TT is that they block
vital functions of the cancer cells while affecting little the healthy tissues. Another aspect of
treatment optimization is patient selection. To that end our better understanding of
molecular biology is pivotal. The recognition of cancer-promoting alterations in the genome
of malignant cells helps not only in the development of targeted therapies but also as
prognostic and predictive biomarkers. A specific example is the gene that encodes for the
protein BRAF. BRAF is a protein that participates in the RAS-RAF-MEK-ERK-MAP kinase
pathway that mediates cellular responses to growth signals. When the gene encoding for
BRAF is mutated, specifically a single substitution missense mutation (V600E), it leads to the
production of a continuously activated protein that has been shown to have a critical role in
82
the development of malignant tumors such as melanoma. The mutated variant of BRAF
(V600E) is detected in 8-10% of patients with CRC as per recent reports, but its role as a
predictive or prognostic biomarker is not yet clear.
The microsatellite instability of DNA (MSI) is characterized by the absence of protein
expression encoded by the corresponding mismatch repair (MMR) genes (hMLH1, hMSH2,
hMSH6). MSI is observed in the vast majority of patients with hereditary non-polyposis
colon cancer and in 20% of patients with sporadic CRC. In the sporadic form the MMR genes
are inactivated due to epigenetic methylation in the promoter region of hMLH1 or by
alteration of the hMSH2 or hMSH6 genes. Microsatellite genotyping of CRC patients is based
on specific standard criteria using specific panels. The use of MSI as predictive and/ or
prognostic biomarker is not sufficiently clarified.
Cyclin D1 is a G protein that with the aid of cyclins D2 and D3 activate the cyclin dependent
kinases 4 and 6. Extracellular signals, such as growth factor receptor activation, influence
cyclin D transcription and translation resulting in mitogenic signaling within the cell cycle
machinery. The deregulation of cyclin expression can lead to uncontrolled proliferation
independent of extracellular stimuli. Cyclin D1 is a well-established oncogene with evidence
suggesting that when amplified or overexpressed can contribute in the development of
breast cancer. The role of cyclin D1 in CRC is less well understood nevertheless, it is
implicated in EGFR signaling a pathway of special interest in patients with CRC.
The aim of the present project is to identify the role of BRAF, MSI and cyclin D1 in the
molecular mechanisms that lead to CRC and investigate their role as potential predictive and
/ or prognostic biomarkers.
METHOD
A total of 144 consecutive patients, with histologically confirmed CRC and available tumor
material for molecular analysis, who were treated in the University Hospital of Heraklion,
between January 2002 and December 2006 were included in the study. The study had all the
necessary formal approvals and patients gave their informed consent prospectively. All
patients had documented metastatic disease and received 5-FU based chemotherapy.
83
Formalin-fixed, paraffin embedded tumor sections were reviewed by an experienced
pathologist to confirm the diagnosis and assess the suitability of the samples for further
analysis. The V600E BRAF mutation was detected with the use of real-time PCR (RT-PCR)
using the allelic discrimination method. For the immunohistochemical staining of the MMR
proteins two antibodies were used, specifically anti-hMLH1, anti-hMSH2 and for cyclin D1
the anti-cyclin D1 antibody. The immunostaining kit used was the UltraVision large volume
detection system AP Polymer. The dilutions of the antibodies were: (a) hMLH1 1:50, (b)
hMSH2 1:50 and (c) cyclin D1 1:25. Positive and negative staining controls were used in all
cases. Microsatellite instability was evaluated in all samples using the five reference markers
of the NCI panel in addition to two markers suggested from alternative panels. PCR, using
single strand conformation polymorphism in a non denaturing environment, was used for
the above mentioned molecular markers. All PCR analyses were repeated twice to confirm
the validity of the results. If &γτ30% of the loci examined showed MSI, the tumor was
classified as MSI-H whereas if ΄&λτ30% of loci displayed MSI then the tumor was labeled as
MSI-L or MSS.
Associations between BRAF mutation status, d-MMR, cyclin D1 expression and baseline
characteristics were assessed using the Fisher’s exact test for categorical variables or logistic
regression for continuous variables. PFS was measured from the date of initiation of first
line chemotherapy until the radiologic confirmation of progressive disease or death. Overall
survival was calculated from date of diagnosis of metastatic CRC until death.
RESULTS
The median age of patients was 64 years and 57% of them were men. Metastasectomy was
performed in 21 (15%) patients. The BRAF V600E mutation and tumors characterized as
MSI-H were 12 (8%) and 22 (15%) respectively. Cyclin D1 was overexpressed in 26 (18%)
patients while it was weakly expressed in 63 (44%) and not expressed in 55 (38%).
The median time from initial diagnosis to the radiological documentation of metastatic
disease was 19.3 months (95% CI 14.6-20.3). Median interval from diagnosis of metastatic
CRC to initiation of chemotherapy was 0.8 months (95% CI 0.5-1.1). All patients received 5-
84
FU based treatment. At the time of the analysis 132 out of the 144 (92%) patients were
deceased – 128 due to progressive CRC.
BRAF mutations were found with increased frequency in tumors characterized as MSI-H as
compared to MSI-L or MSS tumors (45% and 1.6% respectively, p=0.001). In addition the
BRAF mutations were also correlated with cyclin D1 overexpression. Specifically cyclin D1
was overexpressed in 58% of BRAF V600E tumors compared to 14% of BRAF wild-type
tumors (p=0.001).
The median PFS of the patient population enrolled was 9.5 months (95% CI 8.4-10.8) and
the median overall survival was 31.5 months (95% CI 26.4-37.7). Patients with wild type
BRAF had significantly increased survival and PFS compared to patients with BRAF V600E (30
vs 14 months and 9.8 vs 2.7 months respectively). Univariate analysis revealed significant
associations of PFS with undifferentiated tumor histology (p=0.001), BRAF mutations
(p΄&λτ0.001) and no metastasectomy (p΄&λτ0.001). Overall survival was associated with tumor
differentiation (p΄&λτ0.001), BRAF mutations (p΄&λλτ0.0001), no metastasectomy (p=0.03) and
finally the sum of chemotherapy lines the patient received (p=0.02).
In multivariate analysis, BRAF V600E and tumor grade were found to be independent
prognostic factors for reduced PFS (HR 2.8, 95% CI 1.4-5.7, p=0.004 and HR 2.0 95% CI 1.3-
3.2, p=0.001 respectively) and OS (HR 5.3, 95% CI 2.5-11.3, p΄&λτ0.001 and HR 2.6, 95% CI 1.6-
4.4, p΄&λτ0.001 respectively).
Forty-eight patients (33%) were treated with cetuximab, 13 (31%) of them carrying the
mutated allele of KRAS in their primary tumors. Interestingly, KRAS and BRAF mutations
were found to be mutually exclusive. The presence of either mutation was significantly
correlated with decreased PFS (p=0.013) and median overall survival (p=0.003).
DISCUSSION
Our results indicate that patients with CRC who tumors harbor the mutated variant of BRAF
have a significantly lower PFS and OS compared to patients with wild-type BRAF. In addition,
the mutated BRAF is predictive of poor response to cetuximab, an anti-EGFR antibody. In
our study we report a median OS of 31.5 months which is higher compared to other studies.
85
Some of the reasons are: (a) a significant percentage of patients underwent
metastasectomy following first line chemotherapy. Metastasectomy, has been shown to
increase survival and should be pursued when feasible. (b) the majority of the patients in
the study received all three chemotherapeutic drugs in the course of their treatment and
finally (c) 45% of the patients received cetuximab and bevacizumab.
In our study the incidence of BRAF V600E was significantly higher in tumors characterized as
MSI-H or tumors that overexpressed cyclin D1. Given that approximately half of the patients
with MSI-H had mutated BRAF, a predictive biomarker for cetuximab, we hypothesize that
patients with known MSI-H status could possibly benefit from BRAF assessment prior to
treatment with anti-EGFR antibodies.
In summary, patients that have the mutant BRAF represent a population with poor
prognosis. Furthermore, treatment with anti-EGFR antibodies should not be routine in
patients with BRAF mutations. On the other hand, we should note that these results need to
be validated prospectively in randomized trials before applied in clinical practice.
|
Search
