IflI Applications in Oncology
Results
Fig. 1 illustrates a standard curve constructed by plotting the log of tenfold serially diluted CEA expressing colon carcinoma cells (COLM-2) against the respective crossover point (Cp). The slope was -3.66 and the mean squared error "(was
less than 0.5, indicating a good amplification efficiency (>90%) and a precise loglinear relation in the range of mRNA corresponding to 1-105 carcinoma cells,
reflecting a comparable sensitivity to conventional nested-primer RT-PCR with a
wide measuring range. CEA mRNA values for patient samples with unknown concentration were calculated with reference to this calibration curve and representative examples are shown in Fig. 1. No CEA mRNA at significant levels was
detected in peripheral blood from 15 healthy volunteers and primary human cultured mesothelial cells from the omentum, both being major cellular constituents
of the peritoneal washes (data not shown).
Total assay time including pretreatment of the sample (15 min), cDNA synthesis from extracted RNA (90 min) and amplification and subsequent data analysis
(45 min) was approximately 3 h, which is less than half the time required for conventional RT-PCR.
The mean CEA mRNA values for 37 negative control patients consisting of 4
patients with benign disease and 33 with mucosa-confined gastric cancer, considered to be clinically benign, was 0.64±2.54 (SD), ranging from 0 to 10.0. On the
other hand, CEA mRNA values in the peritoneal washes from the Douglas cavity
of patients with synchronous peritoneal metastases ranged from 17.1 to 41,920,
except for two false-negative cases. Based on the control patient levels calculated
from the formula (mean+SD x2) and the lowest value in patients with peritoneal
metastasis, we determined a cut-off level at 10 in the present study. Any samples
with a CEA mRNA value less than 10 was classified as negative.
Quantitative analysis by the LightCycler instrument demonstrated average
CEA mRNA values in peritoneal washes from the Douglas cavity ranging from
0.84 for pTl tumors, 513 for pT2 tumors, 3178 for pT3 tumors and 3459 for pT4
tumors. These values correlated statistically with the depth of cancer invasion
(P
belonging to pTl, pT2, pT3, and pT4 categories, the cytology examination detected free cancer cells in 2%, 5%,41 %, and 8%, and conventional RT-PCR gave positive results in 10%,30%,78%, and 69%, respectively. Real-time RT-PCR with the
cut off value detected 4%, 15%,67%, and 54%, respectively, giving intermediate
detection rates between cytology and conventional RT-PCR. This suggests that
false-positive results found in conventional RT-PCR may be eliminated with
LightCycler PCR.
Moreover, CEA mRNA values for peritoneal washes in synchronous peritoneal
metastasis-positive patients was more than 50-fold higher than in those without
metastasis (data not shown). These results suggest a positive correlation between
CEA mRNA levels in peritoneal washes and prognosis [5].
Results
Fig. 1 illustrates a standard curve constructed by plotting the log of tenfold serially diluted CEA expressing colon carcinoma cells (COLM-2) against the respective crossover point (Cp). The slope was -3.66 and the mean squared error "(was
less than 0.5, indicating a good amplification efficiency (>90%) and a precise loglinear relation in the range of mRNA corresponding to 1-105 carcinoma cells,
reflecting a comparable sensitivity to conventional nested-primer RT-PCR with a
wide measuring range. CEA mRNA values for patient samples with unknown concentration were calculated with reference to this calibration curve and representative examples are shown in Fig. 1. No CEA mRNA at significant levels was
detected in peripheral blood from 15 healthy volunteers and primary human cultured mesothelial cells from the omentum, both being major cellular constituents
of the peritoneal washes (data not shown).
Total assay time including pretreatment of the sample (15 min), cDNA synthesis from extracted RNA (90 min) and amplification and subsequent data analysis
(45 min) was approximately 3 h, which is less than half the time required for conventional RT-PCR.
The mean CEA mRNA values for 37 negative control patients consisting of 4
patients with benign disease and 33 with mucosa-confined gastric cancer, considered to be clinically benign, was 0.64±2.54 (SD), ranging from 0 to 10.0. On the
other hand, CEA mRNA values in the peritoneal washes from the Douglas cavity
of patients with synchronous peritoneal metastases ranged from 17.1 to 41,920,
except for two false-negative cases. Based on the control patient levels calculated
from the formula (mean+SD x2) and the lowest value in patients with peritoneal
metastasis, we determined a cut-off level at 10 in the present study. Any samples
with a CEA mRNA value less than 10 was classified as negative.
Quantitative analysis by the LightCycler instrument demonstrated average
CEA mRNA values in peritoneal washes from the Douglas cavity ranging from
0.84 for pTl tumors, 513 for pT2 tumors, 3178 for pT3 tumors and 3459 for pT4
tumors. These values correlated statistically with the depth of cancer invasion
(P
belonging to pTl, pT2, pT3, and pT4 categories, the cytology examination detected free cancer cells in 2%, 5%,41 %, and 8%, and conventional RT-PCR gave positive results in 10%,30%,78%, and 69%, respectively. Real-time RT-PCR with the
cut off value detected 4%, 15%,67%, and 54%, respectively, giving intermediate
detection rates between cytology and conventional RT-PCR. This suggests that
false-positive results found in conventional RT-PCR may be eliminated with
LightCycler PCR.
Moreover, CEA mRNA values for peritoneal washes in synchronous peritoneal
metastasis-positive patients was more than 50-fold higher than in those without
metastasis (data not shown). These results suggest a positive correlation between
CEA mRNA levels in peritoneal washes and prognosis [5].