Mutations In Peripheral Blood Research Articles

Detection of ras gene mutations in peripheral blood of carcinoma patients using CD45 immunomagnetic separation and nested mutant allele specific amplification.

We developed a sensitive technique of detecting circulating tumor cells in carcinoma patients, using CD45 immunomagnetic separation to isolate epithelial cells in blood samples and specific polymerase chain reaction analysis to identify point mutations of the K-ras gene. The method is based on the fact that the peripheral blood mononuclear cells (PBMC) that express CD45 antigen are trapped with anti-CD45 conjugated supramagnetic microbeads while the carcinoma cells that do not express CD45 antigen are not trapped and pass through the magnetic fields. This method concentrated the number of carcinoma cells 3.3 times. After this separation, the modified method of mutant allele specific amplification was applied and this method was able to ten control carcinoma cells in a background of 107 PBMC. A preliminary clinical study demonstrated that six cases of end-stage carcinoma with K-ras mutations in the primary tumor showed the same mutations in the peripheral blood samples, while two cases without K-ras mutation in the primary tumor and 10 healthy volunteers showed no mutation in the peripheral blood samples. The results suggest that this method may be very useful to detect circulating carcinoma cells in the patient whose primary tumor shows K-ras mutations.

Constitutional RB1-Gene Mutations in Patients with Isolated Unilateral Retinoblastoma

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.

C-Ki-ras mutations in peripheral blood of pancreatic cancer patients: A marker for early tumor metastasis

Purpose : To determine the incidence of circulating minimal malignant clone (CMMC) (harboring c-Ki-ras-2 mutation) in peripheral blood (PB) samples of untreated pancreatic cancer using polymerase chain reactio0n (PCR) analysis of c-Ki-ras-2 oncogene. Methods and Materials : Experiments were carried out in fresh tumor, peritoneal washings (PW), and PB samples of untreated pancreatic adenocarcinoma patients (both resectable and unresectable). Samples were taken from 16 patients with pancreatic adenocarcinoma for the PCR analysis of mutated c-Ki-ras oncogene. Five tumor samples, 15 PB samples, and 3 PW samples were analyzed for point mutation of the c-Ki-ras gene at codon 12. Results : Out of five tumor samples analyzed for c-Ki-ras mutation, four were positive at the 12th codon. Out of total 15 PB samples, nine were positive for the c-Ki-ras point mutation at the 12th codon. All the positive PB samples showed a base substitution from GGT to GAT in the second position of the 12th codon. Out of three PW samples, two showed mutation at the second from GGT to GAT similar to their PB and tumor samples. Conclusion : Our study indicated the presence of the tumor cells (CMMC) in PB of pancreatic adenocarcinoma that can be identified by PCR analysis of c-Ki-ras oncogene. Patients with presence of CMMC in PB and mutation in PW had tumors that responds poorly to treatment.