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Identifier 000383063
Title Εκτίμησης μοριακών παραγόντων σε ασθενείς με μεταστατικό καρκίνο του παχέος εντέρου που έχουν λάβει θεραπεία πρώτης γραμμής και συσχέτιση τους με την ανταπόκριση στην θεραπεία και την επιβίωση
Alternative Title Evaluation of braf mutational status ,microsatellite instability(MSI) and cyclin D1 expression as predictive and/or prognostic biomarkers in patients with metastatic colorectal cancer who have received 1st line treatment with 5FU -based chemotherapy
Author Παπαδάτος-Πάστος Διονύσιος Ι.
Thesis advisor Γεωργούλιας, Βασίλειος
Reviewer Μαυρουδής, Δημήτριος
Τζαρδή, Μαρία
Σαμώνης, Γεώργιος
Χαλκιαδάκης, Γεώργιος
Σταθόπουλος, Ευστάθιος
Σουγκλάκος, Ιωάννης
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.
Language Greek
Subject Colorectal cancer
Cyclin D1 microsatellite instability
Κύκλινη μικροδορυφορική αστάθεια
Ορθοκολικός καρκίνος
Issue date 2012-03-28
Collection   Faculty/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/5/9/f/metadata-dlib-1394004461-436635-25788.tkl Bookmark and Share
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