Our aims were to: 1. Develop a method sensitive enough to identify the presence of mutated k-ras in serum-derived DNA, and 2. Assess the clinical reliability in pancreatic cancer diagnosis of mutated k-ras detection in serum or bile. DNA was extracted from I mL serum (a DNA concentration less than 100 ng/mL are described in healthy subjects) using affinity colums. As positive control for codon 12 k-ras mutation (wild = GGT, mutated=TGT), the DNA extracted from the pancreatic cancer cell line MIA PaCa 2 was used. To identify the presence of mutated k-ras a ME-PCR was made. The first round of amplification was made using the following primers pair: 5'GAATATAAACTIGTGGTAGTIGGACCT3' (KR3) AND 5'TCATGAAAATGGTCAGAGAAACC3' (KR4), the underlined base introducing an artificial restriction site for the enzyme BstO I in wild k-ras. This step was followed by a digestion with BstO I and a further amplification step using the following primers: KR3 and 5'TCAAAGAATGGTCCTGQACC3' (KR2M). The latter primer introduces a second restriction site for BstO I to verify the restriction efficacy of the second digestion. Normal DNA (100 ng) was added with different amounts of mutated DNA (w/m=50/0, 50/1, 100/1, 200/1, 300/1, 400/1, 500/1). We performed 12, 13, 14,24 and 27 amplification cycles with any of the above samples in the first PCR round. Each sample was subjected to the second PCR for 24, 25, 26, 27, 28 and 29 cycles. A total of 200 final results were analyzed. The introduction of an artificial restriction site implies in ME-PCR the development of false positive signals, which were evidenced in some of our negative control samples (w/m = 50/0). The optimal condition to obtain a satisfactory sensitivity (w/m=300/1), associated with the absence of false positive signals, was the following: first PCR: 14 cycles followed by a second PCR of 27 cycles. The analysis of sera obtained from 29 patients with pancreatic cancer demonstrated the presence of mutated k-ras in two cases (6.9%), who had liver metastases. Bile samples were obtained from II patients, and mutated k-ras was found in 4/11 (36%). In conclusion: we optimized a method to extract DNA from I mL of serum; this DNA was demonstrated to be suitable for k-ras amplification and for the identification of codon 12 point mutations. However the identification of mutated k-ras in serum or bile has an unsatisfactory sensitivity in detecting pancreatic cancer and cannot be proposed for clinical routine applications.

K-ras point mutations detection in pancreatic cancer serum and bile-derived DNA.

ZAMBON, CARLO-FEDERICO;FOGAR, PAOLA;GRECO, ELIANA;BASSO, DANIELA;PLEBANI, MARIO;PEDRAZZOLI, SERGIO
2000

Abstract

Our aims were to: 1. Develop a method sensitive enough to identify the presence of mutated k-ras in serum-derived DNA, and 2. Assess the clinical reliability in pancreatic cancer diagnosis of mutated k-ras detection in serum or bile. DNA was extracted from I mL serum (a DNA concentration less than 100 ng/mL are described in healthy subjects) using affinity colums. As positive control for codon 12 k-ras mutation (wild = GGT, mutated=TGT), the DNA extracted from the pancreatic cancer cell line MIA PaCa 2 was used. To identify the presence of mutated k-ras a ME-PCR was made. The first round of amplification was made using the following primers pair: 5'GAATATAAACTIGTGGTAGTIGGACCT3' (KR3) AND 5'TCATGAAAATGGTCAGAGAAACC3' (KR4), the underlined base introducing an artificial restriction site for the enzyme BstO I in wild k-ras. This step was followed by a digestion with BstO I and a further amplification step using the following primers: KR3 and 5'TCAAAGAATGGTCCTGQACC3' (KR2M). The latter primer introduces a second restriction site for BstO I to verify the restriction efficacy of the second digestion. Normal DNA (100 ng) was added with different amounts of mutated DNA (w/m=50/0, 50/1, 100/1, 200/1, 300/1, 400/1, 500/1). We performed 12, 13, 14,24 and 27 amplification cycles with any of the above samples in the first PCR round. Each sample was subjected to the second PCR for 24, 25, 26, 27, 28 and 29 cycles. A total of 200 final results were analyzed. The introduction of an artificial restriction site implies in ME-PCR the development of false positive signals, which were evidenced in some of our negative control samples (w/m = 50/0). The optimal condition to obtain a satisfactory sensitivity (w/m=300/1), associated with the absence of false positive signals, was the following: first PCR: 14 cycles followed by a second PCR of 27 cycles. The analysis of sera obtained from 29 patients with pancreatic cancer demonstrated the presence of mutated k-ras in two cases (6.9%), who had liver metastases. Bile samples were obtained from II patients, and mutated k-ras was found in 4/11 (36%). In conclusion: we optimized a method to extract DNA from I mL of serum; this DNA was demonstrated to be suitable for k-ras amplification and for the identification of codon 12 point mutations. However the identification of mutated k-ras in serum or bile has an unsatisfactory sensitivity in detecting pancreatic cancer and cannot be proposed for clinical routine applications.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2526171
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