الفهرس 
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Abstract
The presence of nucleic acids, DNA and RNA in the plasma of healthy and sick individuals has been known for more than 50 years and specifically in cancer patients since 1948. however, it was not until the classic studies of Stroun et al. (1989) that the neoplastic characteristics of plasma DNA in cancer patients were recognized. The present study was conducted on 59 patients with sporadic CRC (23 females and 36 males), their ages ranged from 22 to 80 years (mean age 46.77 21 years) and eleven control subjects (5 females and 6 males), their mean age was 40.4 11.9. Patients were selected from inpatients of GEC during the period from August 2002 to March 2004. Patients with family history of familial adenomatous polyposis or HNPCC were excluded, they were grouped according to modified Astler Coller of Dukes’ staging. They were subjected to thorough history and clinical evaluation related to their complaint. Routine laboratory investigations were carried out for all subjects. Fasting, morning, venous blood samples were withdrawn preo-peratively and delivered into 2 separate tubes. Serum was separated from the first tube and handled to be used for the special investigations i.e. serum CRP, CEA, CA19-9 and nucleosomes. Plasma was separated from the second tube containing EDTA as anticoagulant, stored at -20oC and used later for DNA extraction. The buffy coat was collected, washed and suspended in a final volume of 1 ml PBS to be used as a negative control for each sample. Results revealed significant increase in circulating nucleosomes in CRC patients compared to controls with highest sensitivity, specificity and excellent discrimination between patients and control, this was followed by CEA (moderate discrimination) and lastly CA19-9 with poor discrimination. Effects of DM, HCV positivity, preoperative blood transfusion and smoking on serum nucleosomes, CEA and CA19-9 concentration were studied. Except for the significant increase in serum nucleosomes concentration in smoker patients there were insignificant increases in all these markers in diabetic, HCV positive and transfused patients. Also, the results of multiple linear regression analysis showed some sort of dependence between CA19-9 and HCV on circulating serum nucleosomes concentration. As regards the relation of pathological features of the tumor and serum nucleosomes, CEA and CA19-9 concentrations, there was an insignificant increase in serum nucleosomes and insignificant decrease of CEA with decreasing grade of tumor differentiation. Also, there was significant increase in serum nucleosomes concentration in patients with poorly differentiated tumors versus patients with well differentiated tumors. Circulating serum nucleosomes levels run parallel with lymph node metastasis while CA19-9 showed insignificant increase with lymph node metastasis. Circulating serum nucleosomes concentration were significantly increased with increasing tumor stage. However, there were insignificant difference in the positivity rate of the 3 markers with Dukes’ staging. Also, a significant positive correlation was observed between serum ionized calcium and circulating nucleosomes concentration. As regards K-ras, among 59 CRC patient samples who were analyzed for codon 12 and 13, only 4 patients (6.7%) showed plasma mutant K-ras bands of 128-134bp. Two cases in stage B1 (13.3%), one case in stage B2 (7.69) and one case in stage C1 (8.33). All on codon 12. they were serine, valine, alanine and arginine in the 4 recorded cases respectively. Conclusions: The detection of K-ras mutation in plasma of CRC patients suggest that they arise from the primary tumor. Circulating levels of nucleosomes are not organ specific marker, with highest sensitivity and specificity for apoptosis that should be applied on a large scale of cancers with respect to clinico-pathological variables that could affect its level before establishing a definite diagnostic value. Group B Dukes’ patients deserve special attention because the detection of K-ras mutation can differentiate aggressive (non responders to chemotherapy) from indolent forms (highly responsive to chemotherapy). Use of CEA and CA19-9 in diagnosis of CRC requires adjustment for the cutoff values of these markers to obtain highest sensitivity and specificity. Use of CA19-9 in diagnosis of CRC deserve second thought because it is expensive, non specific and difficult to be interpreted. The early diagnosis and rapid excision of a small cancerous lesion is highly needed to reduce the pain and suffering of patients who progress to advanced cancers. Our challenge should be to translate new discoveries in cancer genetics promptly from the bench to the bedside. Recommendations: Collection and analysis of more than one sample on different days may increase sensitivity for detection of plasma K-ras mutation in CRC. The sensitivity and specificity of AS-PCR in detecting mutation (malignancy) are encouraging when applied to selected groups of patients with mutant primary tumors. Other techniques such as RFLP could be more sensitive than As-PCR in detection of K-ras mutation as the presence of mutation will prevent cleavage of restriction enzyme. Simultaneous search for distinctly different mutations (p53, DCC) within the same specimen would increase the diagnostic range of plasma DNA testing. Further research, focusing on the association between precancerous lesions (adenoma) and early stages of CRC with appearance of mutant plasma DNA, will help to define its role as a screening, diagnostic and consequently prognostic markers for cancer in the future.