Is the Information Yielded by Detection of Circulating HCC Cells in Peripheral Blood of Clinical Relevance?

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies. Tumor recurrence is, however, frequent and may be due to undetectable tumor cells. Reverse transcriptase polymerase chain reaction (RT-PCR) has been used to detect those tumor cells in peripheral blood but has several disadvantages. METHODS: Peripheral blood from 47 patients (group A, 19 patients with newly diagnosed HCC; group B, 20 control patients with non-HCC liver lesions; Group C, 8 HCC aftercare patients) was tested for the presence of circulating HCC cells by demonstration of alpha-fetoprotein mRNA by the RT-PCR technique. RESULTS: Six patients with newly diagnosed HCC tested positive for circulating HCC cells. No patient in the control group or the aftercare group was positive for alpha-fetoprotein mRNA. CONCLUSIONS: The specificity (100%) of alpha-fetoprotein mRNA as a marker for HCC cells in circulating blood was satisfactory but the sensitivity (31.6%) was disappointing. Our experience is that this method of detecting circulating HCC cells did not provide any clinically relevant information and that we should focus our interest on methods like the isolation by size of epithelial tumor cells for the detection of circulating tumor cells in the future.

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide. Treatment options are severely limited by the frequent presence of metastases. If hepatocyte-specific mRNAs are detected in the circulation, it is possible to infer the presence of circulating, presumably malignant, liver cells. If these can be quantified, it is possible to predict the likelihood of haematogenous metastasis. In this investigation, we have attempted to gain an index of the mass of circulating HCC cells (with reference to the number of hepatoblastoma cells) by measuring the amounts of PCR products for albumin (alb) mRNA and alpha-fetoprotein (afp) mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis. For calibration, total RNA from 1-10(6) HepG2 cells was mixed with total RNA from 10(6) normal peripheral mononuclear cells. A linear relationship was demonstrated between the amount of alb- or afp PCR product and the level of HepG2 total RNA spiked. The assay is sensitive down to a detection level of one HepG2 cell. Alb mRNA was detected in 50% of 18 normal subjects and afp mRNA in only two normal subjects. The alb mRNA cut-off level for the normal was exceeded by seven normal subjects and 34 out of 64 HCC patients, and that for afp mRNA was exceeded by six HCC patients but none of the normal subjects. The level of alb mRNA detected was not linearly proportional to the amount of afp mRNA detected in peripheral blood of the same patients, suggesting heterogeneous expression of alb and afp genes in different circulating tumour cells. In addition, no significant linear association between the levels of afp mRNA and serum AFP was observed. Semiquantification of both mRNA markers for HCC cell detection may prove useful in prediction of metastases.

Detecting AFP mRNA in peripheral blood of the patients with hepatocellular carcinoma, liver cirrhosis and hepatitis

DETECTION OF β-HUMAN CHORIONIC GONADOTROPIN EXPRESSING CELLS BY NESTED REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION IN THE PERIPHERAL BLOOD STEM CELLS OF PATIENTS WITH ADVANCED GERM CELL TUMOR

To detect hepatocellular carcinoma (HCC) cells in the circulating peripheral blood, we previously designed a highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) method targeting alpha-fetoprotein (AFP) messenger RNA (mRNA). Using this method, we analyzed peripheral blood of in- and out-patients bearing HCC for 2 months consecutively and examined the outcome thereafter. All 11 patients with recurrence either in the liver alone or in both the liver and the lung were positive for AFP mRNA. Among 10 recurrence-free patients, 2 patients were AFP mRNA-negative and remained recurrence-free during a period of 22 months observed. Four of 8 AFP mRNA-positive, recurrence-free patients developed a clinically evident recurrence in the liver after 2 to 16 months. Seven of 8 preoperative patients were already positive for AFP mRNA and the remaining negative patient became positive during surgery. Four of 6 preoperatively positive and still-alive patients had recurrence in the liver after 2 to 9 months. None of the HCC-negative patients were positive for AFP mRNA. We actually found an AFP protein-positive cell in peripheral blood obtained from one of the AFP mRNA-positive patients by immunostaining. The present results suggest that circulating HCC cells may have some relationship with the recurrence of HCC in the liver.

Analysis of α-fetoprotein mRNA in peripheral blood: detection and semi-quantitation by reverse transcription polymerase chain reaction

How should patients with hepatocellular carcinoma recurrence after liver transplantation be treated?

This work aimed to investigate the correlation of the expression of α-fetoprotein (AFP) and glypican-3 (GPC3) mRNAs in the peripheral blood with primary hepatocellular carcinoma (HCC) and HCC metastasis by using multiple fluorescence quantitative reverse transcriptase-polymerase chain reaction (FQ-RT-PCR). Peripheral blood samples from 100 patients with HCC were collected. The positive expression rates of AFP mRNA of HCC, hepatitis B, and cirrhosis patients were 56, 5, and 10%, respectively. AFP mRNA was not detected in healthy subjects, hepatic hemangioma, or hepatic metastasis patients' samples. Those of GPC3 mRNA of HCC patients were 76%. GPC3 mRNA was not detected in healthy subjects, hepatitis B, cirrhosis, hepatic hemangioma, or hepatic metastasis patients' samples. In HCC patients' samples, the combined positive rate of AFP and GPC3 mRNA expressions was 81%. The relative expression levels of GPC3 mRNA in the metastasis group and nonmetastasis group were 0.98±0.38 and 0.72±0.26, respectively, and showed significantly different (P=0.001). However, no significant difference was observed in the AFP mRNA expression levels (P=0.134). In conclusion, the sensitivity of HCC diagnosis can be improved by combined detection of AFP and GPC3 mRNA expressions. GPC3 mRNA is HCC-specific, and may indicate HCC metastasis.

Detection of liver cells in peripheral blood of patients with advanced-stage hepatocellular carcinoma

The aim of this study was to establish sensitive techniques for the detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests. For this purpose, we used immunocytochemical staining for cytokeratin filaments and reverse transcriptase-polymerase chain reaction (RT-PCR) for epidermal growth factor receptor (EGF-R) and germ cell alkaline phosphatase (GCAP). Germ cell tumor lines Tera-1 and Tera-2 were titrated into normal peripheral blood. Immunocytochemical staining allowed detection of one cytokeratin-positive tumor cell in 10(5) cells. RT-PCR for EGF-R revealed one tumor cell in 10 cells for Tera-1 and one tumor cell in 10(3) cells for Tera-2, while RT-PCR for GCAP displayed a sensitivity of one tumor cell in 10(6) cells for Tera-1, one tumor cell in 10(4) cells for Tera-2, and no positivity in normal mononuclear cells (n = 20) and progenitor cell harvests (n = 5). The analysis of peripheral blood and progenitor cell harvests from 20 patients with germ cell tumors revealed tumor cell contamination in three patients using immunocytochemical staining and in seven patients by RT-PCR for GCAP. We conclude that RT-PCR for GCAP appears to be suitable for the sensitive detection of residual germ cell tumor cells in peripheral blood and progenitor cell harvests.

Alpha-fetoprotein messenger RNA (AFP mRNA) in the peripheral blood (PB) of patients with hepatocellular carcinoma (HCC) has been considered to represent isolated tumor cells. We investigated its association with the prognosis after curative resection. Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, AFP mRNA in the PB was determined prospectively in control and in 81 patients with curative resection for HCC. Twenty-two (27.2%) and 19 (23.4%) of 81 HCC patients had AFP mRNA in their pre- and postoperative PB. Its presence preoperatively was not associated with an increased risk of HCC recurrence (54.5% vs 40.7%, p= 0.264). In contrast, the postoperative presence associated significantly with a higher incidence of recurrence (89.5% vs 30.6%, p < 0.001), irrespective of preoperative status. The odds ratio for HCC recurrence was 19.2 (95% confidence interval [CI]: 4.0- 91.7). The cmulative probability of recurrence-free survival was also much lower in patients with postoperatively positive AFP mRNA (p < 0.001). The Cox proportional hazards model also demonstrated a significant association with recurrence (p= 0.002). Preoperative serum AFP is also a significant factor and combination with postoperative AFP mRNA enhances the predictability, sensitivity (75.0%), specificity (93.3%), positive prediction (90.0%), and negative prediction (82.4%). The postoperative detection of AFP mRNA in PB is associated with an increased risk of earlier HCC recurrence. Combination with preoperative serum AFP is useful in predictability.

New frontiers in liver resection for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a fatal disease and patients with HCC frequently die from metastasis. The mechanisms of HCC metastasis are not completely understood. In the present study, in vitro and in vivo data showed that HCC cells promoted cancer cell proliferation and lung metastases formation in a paracrinal/endocrinal way. We found that HCC-derived exosomes mediated this phenomenon and observed enhanced cell adhesion in the presence of these malignant exosomes. We further identified that reactive oxygen species (ROS) regulated the adhesive molecules. Intriguingly, attached HCC cells released exosomes containing both SMAD Family Member 3 (SMAD3) protein and mRNA, which were delivered to detached HCC cells and facilitated their adhesion. These exosomes induced enhanced SMAD3 signaling in the recipient HCC cells and increased their adhesive ability. In addition, we showed that SMAD3-abundant exosomes existed in the peripheral blood of patients with HCC, and their levels correlated with disease stage and the SMAD3 expression of primary tumors. Our study suggested a possible mechanism by which primary HCC supported metastases formation and revealed the role of SMAD3 in the exosomes-mediated crosstalk between primary and circulating HCC cells.

Lunx Is a Superior Molecular Marker for Detection of Non-Small Lung Cell Cancer in Peripheral Blood

To detect MAGE-1 mRNA and AFP mRNA in the peripheral blood of patients with hepatocellular carcinoma (HCC) perioperatively, and explore their relationships with recurrence. Peripheral blood from 45 patients with HCC, obtained perioperatively, was tested for the presence of MAGE-1 mRNA and AFP mRNA by nested RT-PCR. All patients were followed for metastatic recurrence for average 11 months after surgery. The peripheral blood from 22 patients with hepatitis B and cirrhosis, 11 patients with hepatic hemangioma, 12 patients with metastatic liver cancer and 20 healthy volunteers served as control. Four of the twelve (33.3%) metastatic hepatic cancer patients were positive for MAGE-1 mRNA, and three of the twenty-two (13.6%) patients with hepatitis B and cirrhosis were positive for AFP mRNA while the other controls kept negative in their peripheral blood. Detection rates for MAGE-1 mRNA/AFP mRNA in the peripheral blood of patients with HCC were 42.2%/51.1% before operation, 20%/24.4% seven days after surgery, and 15.6%/22.2% 28 days later respectively. In chi-square analysis, detection of peripheral-blood MAGE-1 mRNA and AFP mRNA 28 days after surgery indicated metastatic recurrence. Furthermore, the two-marker detection had a higher specificity for prediction of metastatic recurrence than single marker detection. Combined detection of cancer-specific MAGE-1 mRNA and hepatocyte-specific AFP mRNA by nested RT-PCR in the peripheral blood of patients with HCC 28 days after surgery may be useful for the prediction of metastatic recurrence.