Role of circulating tumour cells in predicting recurrence after excision of primary colorectal carcinoma.

BACKGROUND: This study assessed the potential for reverse transcriptase-polymerase chain reaction (RT-PCR)-based circulating tumour cell identification to predict colorectal cancer recurrence. METHODS: mRNA for carcinoembryonic antigen and cytokeratin 20 was identified by RT-PCR in blood from patients with colorectal cancer, before and after primary tumour resection. Cancer recurrence was assessed at follow-up, and the accuracy of RT-PCR and primary tumour lymph node positivity in predicting recurrence was estimated. RESULTS: One hundred and ninety-six patients with colorectal cancer were studied over a median follow-up of 1393 days from surgery. Regression analysis selected 24-h post-resection RT-PCR positivity (hazard ratio for a positive test in predicting recurrence 8.66 (95 per cent confidence interval (c.i.) 3.08 to 24.33)) before lymph node involvement (hazard ratio 7.92 (95 per cent c.i. 3.26 to 19.20)). When 24-h post-resection RT-PCR was combined with lymph node positivity, the hazard ratio increased to 18.54 (95 per cent c.i. 4.01 to 85.11), attributing a 3 per cent recurrence risk to 52 per cent, and a 50 per cent recurrence risk to 48 per cent, of patients with colorectal cancer resected with curative intent. CONCLUSION: RT-PCR positivity within 24 h of primary colorectal cancer resection is a strong predictor of colorectal cancer recurrence, and may be useful clinically.

P53 mutation and response to hepatic arterial floxuridine in patients with colorectal liver metastases.

PURPOSE: To establish the relationship between the number and site of p53 genomic mutations in metastatic colorectal cancer, and the response to hepatic arterial floxuridine. METHODS: Liver metastasis biopsies were collected, at the time of laparotomy for hepatic arterial cannulation. in 28 patients with metachronous colorectal liver metastases. p53 Gene mutations were assessed using reverse transcription, nested polymerase chain reaction, single strand conformational polymorphism and gene sequencing. Chemotherapy response was determined from computerised liver tomograms after 4 months of treatment. RESULTS: Liver metastasis p53 mutation was identified in 21 (75%), and p53 "hot spot" mutation in 11 (39%) patients. There was a significantly lower prevalence (Fisher's, P=0.001) of patients with p53 "hot spot"-mutated liver metastases in stable disease and partial response (5/22) than in progressive (6/6) disease groups. Significantly fewer (Mann-Whitney U, P=0.002) p53 "hot spot" mutations/biopsy were observed in liver metastases from stable disease and partial response (median 0, iqr. 0-0) than in progressive (median 1, iqr 1-2) disease patients. p53 "Hot spot"-mutated liver metastases were associated with significantly shorter survival times (logrank P=0.05) after hepatic arterial floxuridine. Significant response or survival-time differences by total or L2/L3 zinc-binding site p53 mutations were not detected. CONCLUSIONS: The results support a role for p53 "hot spot" mutations in colorectal liver metastasis resistance to fluorinated pyrimidines, and suggest that the presence of such mutations may be a contra-indication to treatment of colorectal liver metastases with hepatic arterial floxuridine.

Relationship between tumour vascularity and circulating cancer cells in patients with colorectal carcinoma.

BACKGROUND: Colorectal cancer vascularity correlates with risk of metastasis. Greater tumour vascularity may increase haematogenous dissemination by providing a larger vessel area for tumour cell invasion into the circulation. We assessed whether the prevalence of tumour cells in the circulation of colorectal carcinoma patients (CTC) increased with tumour vascularity. METHODS: Pre-operative blood samples were assessed for circulating tumour cells using RT-PCR for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) mRNA. Vessel count and volume were morphometrically assessed from tumour biopsies after vasculature staining. RESULTS: Thirty-three colorectal cancer patients (M:F, 20:13; mean age 66 years, SD 11 years) were studied. One or more blood samples were RT-PCR positive for either CEA or CK20 mRNA or both, in 28 (85%) patients. There were no significant differences in the prevalence of RT-PCR positive patients between high and low tumour vascularity groups, or in tumour vessel counts or volume in RT-PCR positive compared with negative patients. CONCLUSIONS: These results do not support vascularity related variation in access of tumour cells to the circulation as an explanation for the correlation between tumour vasculature and metastasis. Tumour vascularity and metastatic potential may be linked phenotypes rather than cause and effect.

P53 mutations in primary and metastatic tumors and circulating tumor cells from colorectal carcinoma patients.

Circulating tumor cells could provide a relatively noninvasive and repeatable source of information about tumor cell genotype that might influence treatment and estimation of prognosis. We developed a technique for identifying p53 mutations in tumor cells isolated from the peripheral venous blood of colorectal cancer patients and compared the prevalence and position of these mutations with multiple solid tumor samples from the same patient. We used immunomagnetic beads to isolate tumor cells, reverse transcriptase-nested polymerase chain amplification of the coding region between exons 4 and 9 within the p53 gene, and automated gene sequencing. Nineteen p53 mutations were detected in solid tumor samples from 19 of 41 colorectal carcinoma patients. An identical p53 mutation was invariably present in all samples from primary and metastatic colorectal tumor biopsies within the same patient. p53 mutations were detected in peripheral blood from 8 of these 19 patients with p53-mutated solid tumors. Where identified, the pattern of mutation in peripheral blood samples was invariably the same as in matching solid tumor samples. A single colorectal carcinoma biopsy provided reliable p53 gene mutational information in colorectal carcinoma. Detection of this p53 mutation in tumor cells from peripheral blood was achieved with an approach based on cell selection for epithelial characteristics, reverse transcription-PCR, and gene sequencing.

Plasma vascular endothelial but not fibroblast growth factor levels correlate with colorectal liver mestastasis vascularity and volume.

The extent to which plasma levels of angiogenic factors in healthy individuals and tumour volume-related variations in colorectal cancer affect the accuracy of circulating angiogenic factors as predictors of colorectal cancer vascularity is unknown. We used enzyme-linked immunosorbant assay to measure plasma vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels in colorectal liver metastasis (CLM) patients, and 'no cancer' controls. CLM volume was determined from computerized tomography scans, and tumour vessel count and vessel volume from anti-endothelial antibody-stained biopsies. There was a significant (P= 0.03) increase in plasma VEGF level in 29 CLM patients (median 180.3 pg/ml(-1), iqr 132.5-284.8 pg/ml(-1) compared with 19 controls (median 125.8 pg/ml(-1), iqr 58.2-235.9 pg/ml(-1). There were significant correlations between plasma VEGF and tumour vessel count (r = 0.66, P = 0.03), tumour vessel volume (r= 0.59, P = 0.03), and CLM volume (r= 0.53, P = 0.03). A VEGF level in the upper quartile of the plasma VEGF distribution had a 70% sensitivity and 75% specificity in predicting an upper quartile liver metastasis tumour vessel count. No relation was identified between CLM and plasma bFGF levels. Plasma VEGF level predicted CLM vascularity, despite an overlap with normal levels and tumour volume-related variations.

Venesection needle coring increases positive results with RT-PCR for detection of circulating cells expressing CEA mRNA.

We assessed whether circulating cell positivity using RT-PCR for carcinoembryonic antigen (CEA) cDNA was affected by venesection via a needle compared with a pre-aspirated venous cannula, and by increased PCR cycles. Systemic blood was sampled by needle and pre-aspirated cannula in 101 healthy individuals with no cancer history. After erythrocyte removal, samples were subjected to RT-PCR using specific primers for CEA, with 29 or 35 RT-PCR cycles. There was a significant difference between the number of subjects whose samples were negative when collected via needle venesection and positive when collected via pre-aspirated cannula, compared with positive by needle venesection and negative by pre-aspirated cannula for both 29 (P = 0.016) and 35 (P = 0.0111) RT-PCR cycles. Venesection technique (P = 0.01) and number of cycles (P = 0.003) were significant predictors of a positive result. Positive results in healthy subjects were reduced to less than 3% when an aspirated cannula was used for venesection and >29 PCR cycles were avoided.

Increased detection of circulating tumor cells in the blood of colorectal carcinoma patients using two reverse transcription-PCR assays and multiple blood samples.

The objectives of this study were to assess whether the use of two reverse transcription-PCR (RT-PCR) cDNA assays and multiple blood sampling increased circulating tumor cell detection in colorectal cancer patients. Systemic blood was sampled three times at 1-min intervals in 100 colorectal cancer patients (50 primary tumors only and 50 liver metastases), and in 70 control patients without known cancer. After removal of the erythrocytes, samples were subjected to separate RT-PCR reactions using specific primers for carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20). Statistical analysis was performed by the two-sample binomial test and the one-sided McNemar test. There were significant increases in circulating tumor cell positivity when CEA and CK20 assays were used together as compared with either CEA or CK20 assay used alone. There were also significant increases in circulating tumor cell positivity for either CEA or CK20 assay used alone when the results from two blood samples were compared with the results from one sample. Circulating colorectal cancer cell positivity rose from 48% (CEA) and 34% (CK20) with one assay of one sample to 74% when both assays of three samples were used to identify circulating tumor cells. Three non-cancer control patients (4.3%) were positive for either CEA (two patients) or CK20 (one patient). Tumor cells were identified more frequently in the circulation of colorectal cancer patients than had been suggested previously. RT-PCR-based studies of the clinical significance of circulating cancer cells in colorectal cancer should involve multiple blood samples with identification of multiple tumor-related cDNA products.

Detection of colorectal cancer cells in peripheral blood by reverse-transcriptase polymerase chain reaction for cytokeratin 20.

The staging of colorectal cancer currently depends on pathological examination of the surgical specimen and regional lymph nodes, accompanied by imaging tests such as computed tomography (CT) scanning. However, alternative molecular methods to detect circulating tumour cells in blood or bone marrow may provide additional information about the extent of disease and prognosis. We have previously reported the development of a reverse-transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 20 (CK 20) mRNA to detect circulating epithelial tumour cells. In this study, we report on the application of this method for detecting circulating tumour cells in patients with colorectal cancer. Using this method, CK 20 mRNA was detected in 8/8 human colorectal cancer cell lines, in 8/9 biopsies from primary colorectal tumours and in 9/10 biopsies of liver metastasis in patients with metastatic colorectal cancer, suggesting that CK 20 may be a useful target for the detection of circulating tumour cells in this patient group. In spiking experiments, 10 cells were consistently identified in 2 ml of whole blood (1 x 10(6)-1 x 10(7) mononuclear cells). In 12/25 (48%) peripheral blood samples from patients with known metastatic colorectal cancer, CK 20 mRNA was detected. However, there was no correlation between the detection of CK 20 mRNA in the peripheral blood and disease progression and survival in this group of patients. CK 20 mRNA was detected in 1/12 normal blood samples, which raises questions about the absolute specificity of CK 20 expression.

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia.

The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected].

Modification of the in vitro cytotoxicity of hydrogen peroxide by iron complexes.

The effect of a range of iron chelates on the cytotoxicity of H2O2 was studied on a mammalian epithelial cell line. Iron complexes which were internalised enhanced the cytotoxicity of H2O2 measured by delayed thymidine incorporation. Iron complexed to 8-hydroxyquinoline (Fe/8-HQ) potentiated the cytotoxicity of 50 microM by 38% and Fe/dextran by 23%. Pre-exposure of cells to Fe/dextran at 4 degrees C did not result in any potentiation of H2O2-induced cytotoxicity which we ascribe to failure of the Fe/dextran to be endocytosed at low temperature. Iron complexes which are slowly taken up or remain extracellular protected the cells from H2O2-induced cytotoxicity. Thus, Fe/EDTA inhibited the cytotoxicity of 50 microM H2O2 by 33%; Fe/ADP by 80% and Fe/ATP by 88%, suggesting mutual extracellular detoxification.

The effect of ligands on the uptake of iron by cells in culture.

Uptake of iron by a mammalian epithelial cell line (CNCM I-221) was shown to be dependent on the nature of the iron complex. Iron uptake was demonstrated by cytochemical staining and determination of redox-reactive iron in cell lysates. Three classes of ligands were investigated: (i) low molecular weight hydrophilic compounds, represented by ethylenediamine-tetraacetic acid (EDTA) and other charged ligands such as adenosine phosphates (ATP, ADP, AMP) and diethylenetriaminepentaacetic acid (DTPA), (2) low-molecular weight lipophilic ligands such as 8-hydroxyquinoline (8-HQ) and (3) a high molecular mass ligand, dextran. Iron complexed to 8-HQ accumulated intracellularly, the uptake rate of iron being 4.16 fmoles cell-1 h-1 of exposure at 37 degrees C or 3.86 fmoles cell-1 h-1 at 4 degrees C. Iron-dextran was endocytosed and retained in phagosomes. The uptake rate of iron following exposure to iron dextrans was found to be 5.6 fmoles cell-1 h-1 of exposure at 37 degrees C. In contrast to iron/8-HQ, uptake of iron dextran by cells was inhibited at 4 degrees C. Iron complexed to low molecular weight hydrophilic ligands was not taken up by cells. Cytotoxicity was measured by reduction of plating efficiency or tritiated thymidine incorporation. These tests showed that toxic effects of added iron were demonstrable only in cells exposed to the complex with 8-HQ.

Hydrogen peroxide cytotoxicity. Low-temperature enhancement by ascorbate or reduced lipoate.

The principal mechanism of H2O2 toxicity is thought to involve the generation of hydroxyl (HO.) radicals through its interactions with Fe2+ ions by the Fenton reaction. Of particular interest has been the demonstration by Ward, Blakely & Joner [(1985) Radiat. Res. 103, 383-392] that the cytotoxicity of H2O2 is diminished at low temperature. We have now examined this phenomenon further with a mammalian epithelial cell line (CNCMI-221). Resistance of these cells to 100 microM-H2O2 added extracellularly exhibits a transition in the temperature range between 27 degrees C and 22 degrees C. We have found that the low-temperature resistance to cytotoxic concentrations of H2O2 is abolished by preincubation of cells with reductants such as ascorbate or reduced lipoic acid. This implies that the low-temperature resistance to H2O2 cytotoxicity may be due to inhibition of cellular reductive processes. The restoration of the cytotoxic action of H2O2 at 4 degrees C by ascorbate is prevented by pre-exposure of cells to desferrioxamine. This is evidence that transition-metal ions (such as iron ions) are involved in the cytotoxicity and is consistent with a mechanism of cell damage that depends on the Fenton reaction and a metal ion in the reduced state. Restoration of H2O2 cytotoxicity at low temperature by ascorbate is consistent with the artificial production of an intracellular reducing environment that at normal temperatures is sustained by cellular metabolism.

Obstructive jaundice. Diagnostic and therapeutic considerations.

The diagnostic approach to obstructive jaundice must be individualized on the basis of the sensitivity and specificity of the various procedures available. No single diagnostic approach is optimal in all patients. The physician must be guided by knowledge of the yield, complications, and cost of the tests available. In addition, interventional endoscopic and radiologic techniques add a new therapeutic dimension to the standard surgical approach.

Effect of antibiotic prophylaxis in percutaneous endoscopic gastrostomy.

Thirty-three patients completed a prospective double-blind, randomized study to compare the effect of antibiotic prophylaxis or placebo on percutaneous endoscopic gastrostomy-associated wound infections. We define wound infection and arrive at an incidence of 29.4% in patients receiving Cefoxitin antibiotic prophylaxis and 31.2% in patients receiving placebo. Based on these results, we do not recommend antibiotic prophylaxis for percutaneous endoscopic gastrostomy tube placement.