Where the work of others has been used, it has been duly acknowledged in the text. The tumor caused a narrowing of the esophagus seen in the image shown above.
Adenocarcinoma of the oesophagus
Prognosis, therapeutic and diagnostic strategies: an overview
Computed to mo graphic scanning
Thoracoscopic and laparoscopic staging studies are accurate in identifying lymph node metastases (Krasna et al., 1996 and Luketich et al., 1997). Positron emission tomography (PET) is being investigated as a diagnostic tool as an adjunct to what is currently used. Studies to date have been able to detect previously unsuspected metastatic cancer in nearly 20% of patients previously considered candidates for surgical resection (Block et al., 1997 and Luketich et al., 1997).
The oesophageal cancer tumour staging system
Mlb Non-regional lymph nodes and/or other distant metastasis Tumors of the mid-thoracic esophagus.
Oesophageal cancer by stage
Human papilloma virus infection
It is already known that IIPV types 16 and 18 represent the main risk associated with cervical cancer (Munoz et al., 2003). With regard to OC, these reports present a mixed picture regarding the risk associated with HPV.
Conditions that predispose patients to OSCC development. ................................. 1 9
- Achalasia
People with a history of head and neck cancer are more likely to have OSCC because both conditions are strongly associated with tobacco and alcohol use (Enzinger and Mayer, 2003). In a report where OSCC had previously been unsuspected, 2% of head and neck cancer patients were found to have the disease (Erkal et al., 2001.
The molecular basis for the development of cancer: an overview
Clonal evolution
Mutations that would increase the overall rate of mutations may be responsible for the type of mutation rate observed in tumor cells (Loeb et al., 2003). These SNPs are the result of DNA mismatch repair enzymes that themselves cause loss of function (Naidoo et al., 2005).
The process of carcinogenesis
This in turn can lead to downstream inactivation of genes (Nelson et al., 1992, Minamoto et al., 1999). Moreover, the promotion is also reversible, as removal of chemical agents can lead to preventing or reducing the frequency of tumor formation (Iwamoto et al., 2000).
Epigenetics and cancer
DNA regions in the 5' promoter regions of almost 50% of known human genes are said to contain the so-called Ultimately, the genes associated with these methylated sites remain inaccessible to transcription factors, etc., resulting in "epigenetic silencing" of the gene.
The molecular basis of oesophageal squamous cell carcinoma development
- The role of tumour suppressor genes in oesophageal squamous cell carcinoma
- p53 37
- Apoptosis-related genes
- Oncogenes and oesophageal squamous cell carcinoma
- Microsatellite alterations in human cancer
In particular, Ramburan et al. 2004) found that multiple abnormalities of the DCC locus were involved in the progression of nephroblastomas. The location of the microsatellite marker would likely be an area of tumor suppressor activity (Oda et al., 1997).
The cell cycle theory
- The Cyclin-Cdk complexes
- The pRb/E2F pathway
- The Cip/Kip thmily of Cdk inhibitors 56
- p?1 Cip :RAF/ an d p 1 7 ,Lopr and their roles in cancer
Towards the late GI phase of the cell cycle, a key area of cell cycle regulation emerges and is known as the restriction point (Polyak et al., 1994). The D-type cyclins consist of D1, D2 and D3 and have been shown to be active in the G1 and GI - S phase transitions of the cell cycle (Shackelford et al., 1999).
Ethical Approval
DNA isolation
Preparation of paraffin wax-embedded tissue sections for DNA isolation -
Phenol-Chloroform extraction
200 µl of DNA tissue lysis buffer (Appendix Al) was added to each of the tubes, followed by 20 µl of Proteinase K (20mg/m1), The sample tubes were then sealed with ParafilmTM and incubated overnight at approx. 60cC. The supernatant was removed by careful pipetting and the remaining pellet was washed with 75 µl of 80% ethanol (Appendix A4). After a centrifugation step of 3 min at maximum speed, the supernatant was carefully removed and the pellet was allowed to air dry completely to remove any last traces of ethanol.
A 1:100 dilution of each sample was read at a wavelength of 280 nm and the results calculated in concentration units of pg/ml.
Assessment of DNA quality - Insulin PCR
Agarose gel electrophoresis
Annotation and labeling of DNA samples for identification
- Selection of microsattelite markers that flank genes of interest
- Online Public Genome Resources
- Design and construction of primer sequences
- Preliminary PCR test run
- MgC12 optimization
- Template concentration optimisation
- Annealing temperature optimisation
The primer sequence sets for each of the six microsatellite markers, as well as their amplified oligonucleotide lengths and linked genes, are tabulated in Appendix B. For each marker, one sample pair (normal and tumor) was amplified under standard PCR conditions as a preliminary test of PCR viability. This was followed by a final extension step of 72°C for 10 minutes, The reaction was then held at 4°C.
Concentrations of 1\4:2- 1.5, 3, and 5 mM were evaluated, and the reagents used for the reaction as well as the PCR reaction conditions were the same as those listed in section 2.7.4 above, with the exception of the reaction buffer used PCR. The optimal template concentration was determined for each marker (one sample per marker) by varying the amount of DNA template added. The optimal annealing temperature for each marker was determined by performing a PCR reaction using annealing temperatures ranging from 500 to 55 °C.
Preparation ofpolyacrylamide sequencing gel
Gel Cassette cleaning, assembly and loading
The data (Table 7 above) were analyzed with the results obtained from each of the six. Esophageal squamous cell carcinoma can be detected by microsatellite analysis in tumor and serum. Mutational analysis of the human cycle-independent kinase inhibitor p27kipl in primary breast carcinomas.
Frequent loss of heterozygosity in the region, including BRCA I on chromosome 17q irl squamous cell carcinomas of the esophagus. DNA hypermethylation is a mechanism leading to the loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas. Loss of 17p, mutation of the p53 gene and overexpression of p53 protein in esophageal squamous cell carcinomas.
Attachment of gel cassette to the ALFexpress'm DNA sequencer
Sample preparation
Gel loading
The frequencies of LOH-AI were calculated as a percentage of the number of informative cases (1*). Of the 84 cases that had valid entries for the presence/absence of lymph node metastases, 56% were. Decreased levels of the cell cycle inhibitor p27Kip1 protein:. prognostic implications in primary breast cancer.
Increased expression of P27KIP1 protein in human esophageal cancer cell lines overexpressing cyclin D1. Protein expression of the cell cycle inhibitor p27Kipl in malignant melanoma: inverse correlation with disease-free survival. Amplification and expression of TGF-alpha, EGF receptor and c-myc genes in four lines of human esophageal squamous cell carcinoma.
Two CLP1/WAF1 gene variants co-occur and are associated with human cancer. Expression of the cell cycle inhibitor p27K1P1 is a novel prognostic marker associated with survival in epithelial ovarian tumors. Role of the ubiquitin-proteasome pathway in regulating the abundance of cyclin-dependent kinase inhibitor p27.
Expression of p53 and bcl-2 and response to preoperative chemotherapy and radiotherapy for locally advanced squamous cell carcinoma of the esophagus.
Run conditions used on the ALFexpress'TM sequencer
Fragment analysis
Statistical analysis
Light microscopic evaluation
Insulin PCR and agarose gel electrophoresis
Microsatellite PCR
- Preliminary PCR test run
- MgCl 2 optimization
- Template concentration optimization
- Annealing temperature optimization
Regarding the latter, no significant difference was observed between these two concentrations in terms of multiplication. It was decided to use the m1 concentration as this allowed for sufficient amplification using the smallest possible amount of template. 50 °C and 53 °C the primers did not bind strongly enough to the sample strand and non-specific amplification occurred.
Interpretation of data
The results show no significant association between patient age and microsatellite aberrations for any of the markers (p>0.05). Two of the markers used in this study, D12S391 and D I2S358, were also part of their analyses. Crosstab analysis for this marker suggests that there may be an association with poorly differentiated (PD) tumors (Appendix E2, Section 1).
Decreased expression of the cell cycle inhibitor p27Kipl in non-small cell lung carcinoma: a Ras-independent prognostic factor. Nuclear accumulation of p53 significantly correlates with clinical features and inversely with expression of cyclin-dependent kinase inhibitor p21 (WAF1/C1P1) in pancreatic cancer. Formation and activation of the cyclin E-cdk2 complex during phase 01 of the human cell cycle.
I Ifomozygous, no change (11)
- No allelic imbalance (NAI)
- Loss of Heterozygosity-Allelic Imbalance (LOH-AI)
- Microsatellite Instability (MS1)
Overall results of microsatellite PCR
The results of the LOH-AI/NISI analysis for the 96 OC cases are shown in Tables 5 and 6 below. The data in Table 6 shows the results for LOH-AI and MSI recorded for markers near the p2. Statistical analysis was performed using the data available at the time of study.
Statistical Analysis
- Descriptive statistics of categorical and clinico-pathological data
- Age 89
- Tumour stage
- Tumour grade
- TNM staging 9
- Outcome
- Survival time
- Trend analysis
- Kruskal-Wallis test for age vs. marker genotypes
- Chi-squared test and cross tabulation
Most of the tumor stages investigated in this study consisted of stage IIA (47%) and stage III (41%). For more information on different tumor stages, see the section on tumor staging and staging (Chapter 1). Regarding patient outcome, it was recorded that by the end of the study period, 73% of patients were alive and 27% had died.
A Kruskal-Wallis test was performed for age of patient versus the microsatellite abnormalities recorded for each marker. Chi-square tests were performed for all microsatellite markers, regarding patient sex, tumor stage, tumor grade, lymph node metastases, TNM staging, and outcome. Crosstabs for each marker and clinical feature are given in Appendices El and E2, along with the p-values of statistical significance.
Figure 21 (c) and (d) illustrates markers D12S320 and D12S391 where an increased frequency of LOH-Al is associated with older patients. A search of the available literature to date shows that patient age has not been significantly associated with microsatellite aberrations in OSCC (Flu et al., 2005; Kubo et al., 2005; Liu et al., 2005). These researchers concluded that LOH was strongly associated with primary tumors as well as metastatic disease.
Marker D12S391, as shown in Figure 29, revealed that MSI is associated with a reduced time for survival after surgery. For marker D12S364, cases scored as LOH-AI were significantly associated with reduced survival time. Those researchers concluded that although clinical follow-up was limited, increased chances of survival were still associated with fewer microsatellite changes.
The risk of ambiguity in the interpretation of the Kaplan-Meier plots is therefore minimal. In a study on OSCC, Nie and coworkers (2001) investigated DNA hyphenation as a mechanism for loss of expression of the HLA class I genes located at 6p21. This will allow a more accurate assessment of the exact nature of the relationship between D6S1575 and tumor grade.
Allelic imbalance and microsatellite instability of the DCC gene in colorectal cancer in patients under 35 years of age using fluorescent DNA technology.]. Induction of esophageal carcinogenesis by diethylnitrosamine and assessment of the promoting effect of ethanol and N-nitrosonornicotine: experimental model in mice. Clinical outcomes of transhiatal esophagectomy for carcinoma of the lower thoracic esophagus according to biological markers.
Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Aberrant methylation of estrogen receptor and E-cadherin 5' CpG islands increases with malignant progression of human breast cancer.
