0
We're unable to sign you in at this time. Please try again in a few minutes.
Retry
We were able to sign you in, but your subscription(s) could not be found. Please try again in a few minutes.
Retry
There may be a problem with your account. Please contact the AMA Service Center to resolve this issue.
Contact the AMA Service Center:
Telephone: 1 (800) 262-2350 or 1 (312) 670-7827  *   Email: subscriptions@jamanetwork.com
Error Message ......
Original Article |

Detection of Isolated Disseminated Tumor Cells in Bone Marrow and Blood Samples of Patients With Hepatocellular Carcinoma FREE

Peter Kienle, MD; Jürgen Weitz, MD; Rüdiger Klaes, MD; Moritz Koch; Axel Benner, PhD; Thomas Lehnert, MD; Christian Herfarth, MD; Magnus von Knebel Doeberitz, MD
[+] Author Affiliations

From the Department of Surgery (Drs Kienle, Weitz, and Herfarth), the Divisions of Molecular Diagnostics and Therapy (Drs Klaes and von Knebel Doeberitz and Mr Koch) and Surgical Oncology (Dr Lehnert), University of Heidelberg, Heidelberg, Germany; and Central Unit Biostatistics, German Cancer Research Center, Heidelberg (Dr Benner).


Arch Surg. 2000;135(2):213-218. doi:10.1001/archsurg.135.2.213.
Text Size: A A A
Published online

Background  Patients with hepatocellular carcinoma (HCC) often develop recurrences after curative resection or liver transplantation. Therefore, tumor cell dissemination must have occurred preoperatively or intraoperatively. Current staging methods cannot reliably detect micrometastasis. Reverse transcription–polymerase chain reaction (RT-PCR) for α-fetoprotein (AFP) has been used to detect circulating liver cancer cells, but results in blood samples have been contradictory.

Hypothesis  AFP–RT-PCR is a specific and sensitive assay for the detection of disseminated tumor cells in central venous blood and bone marrow samples of patients with HCC and has prognostic relevance.

Design  Prospective consecutive series.

Setting  University hospital.

Patients and Methods  We performed preoperative, intraoperative, and postoperative analyses of central venous blood samples and preoperative analysis of bone marrow samples of patients with HCC and patients without malignant disease, using a modified AFP–RT-PCR method. Preoperative serum AFP levels were measured. Clinical follow-up ranged from 4 to 20 months.

Main Outcome Measures  Sensitivity and specificity of AFP–RT-PCR, correlation of AFP–RT-PCR results to tumor stage and tumor recurrence.

Results  In serial dilution experiments, 50 AFP-expressing HepG2 cells were detected in 10 mL of blood. Peripheral blood samples of 20 healthy volunteers and bone marrow samples of 21 patients with benign diseases consistently tested negative for AFP, whereas 4 of 24 patients with HCC showed AFP expression in bone marrow samples. All these patients had advanced disease; however, correlation of positive RT-PCR results to tumor stage was not significant (P = .07). One of the 4 AFP-positive patients developed an intrahepatic recurrence soon after liver transplantation. Central venous blood of patients with HCC (n = 24) and patients with benign liver diseases (n = 13) equally demonstrated AFP-expressing cells. There was no correlation of RT-PCR results to serum AFP levels.

Conclusions  Perioperative screening for micrometastasis in bone marrow of patients with HCC is sensitive and specific with AFP–RT-PCR and may have prognostic relevance. α-Fetoprotein is not a suitable marker for the detection of tumor cells in central venous blood samples.

Figures in this Article

THE PROGNOSIS of patients with hepatocellular carcinoma (HCC) remains unsatisfactory. Even after complete removal of the tumor either by partial liver resection or by total hepatectomy and following orthotopic liver transplantation, patients often develop intrahepatic or extrahepatic recurrences. Therefore, tumor cell dissemination must have occurred either before or during surgery. Identification of these inapparent micrometastases, which currently cannot be detected by routine staging methods, could facilitate a more accurate preoperative prognosis and possibly allow better selection of patients for different treatment options. Considering the lack of organ donors for liver transplantations and the overall morbidity of the operation, it seems essential to rule out disseminated micrometastatic disease preoperatively.15 Molecular biological techniques have improved the rate of detection of occult tumor metastasis, especially reverse transcription (RT), and subsequent amplification of specific messenger RNA (mRNA) of marker genes has been applied in this context.68 Two such markers have been proposed for the detection of circulating malignant liver cells. Albumin was shown to yield low specificity as albumin transcripts were also detectable in blood samples of a large number of healthy controls, therefore rendering it unsuitable as a marker for tumor dissemination.912 Results for detecting α-fetoprotein (AFP)-expressing cells in peripheral blood samples have been more promising, although here too specificity remains a problem.1317 Patients with acute and chronic hepatitis and liver cirrhosis without malignant disease have also tested positive for AFP mRNA in peripheral blood samples, thereby questioning the validity of the method.18 Especially in the context of surgical treatment of HCC, the detection of AFP mRNA in blood samples appears to be nonspecific and may therefore be unsuitable for prognostic staging and therapeutic decisions.19 Several reports have, however, shown that the detection of micrometastasis of other solid tumors (as demonstrated for breast, colorectal, and lung cancer) in bone marrow samples can be an important prognostic factor with high specificity.2026 The presence of micrometastasis in bone marrow samples of patients with HCC has not yet been investigated. We have therefore established a nested RT–polymerase chain reaction (PCR) system for AFP and tested bone marrow and central venous blood samples of 24 patients with HCC and 21 patients without malignant disease for expression of AFP mRNA.

BONE MARROW AND BLOOD SAMPLES

For bone marrow samples, 10 mL of bone marrow of the iliac crest of both sides was aspirated after bone marrow puncture; blood samples (10 mL) were obtained through a central venous catheter positioned in the superior vena cava or right atrium and all samples were diluted with 10 mL of phosphate-buffered saline. After density centrifugation through Ficoll-Paque (Pharmacia Biotech, Freiburg, Germany) for 30 minutes at 400g, mononuclear cells were harvested from the interphase and washed twice in phosphate-buffered saline. The cell pellet was then shock-frozen in liquid nitrogen and stored at −80°C.

CELL LINE AND SERIAL DILUTION EXPERIMENTS

The hepatoblastoma cell line HepG227 was cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum. Cell counting was performed in a hemocytometer. Serial dilutions of HepG2 cells with phosphate-buffered saline were then added to 10 mL of blood from healthy donors (corresponding to approximately 108 peripheral mononuclear cells) and Ficoll-Paque density centrifugation was performed as described above.

RNA EXTRACTION

Total RNA from bone marrow, blood, cell lines, and frozen tissue sections of tumor and normal liver tissue was extracted using a commercially available RNA isolation system (TRI-Reagent, Molecular Research Center, Cincinnati, Ohio) in accordance with the recommendations of the manufacturer. To eliminate contaminating DNA within the RNA preparation, 1 µg of RNA was digested with ribonuclease-free deoxyribonuclease I for 15 minutes at 25°C as recommended by the supplier (Life Technologies Inc, Gibco BRL, Karlsruhe, Germany).

RT-PCR ANALYSIS

Sequences for oligonucleotide primers in the AFP complementary DNA (cDNA) sequence28 were identified through the Heidelberg Unix Sequence Analysis Software Program (German Cancer Research Center, Heidelberg). A nested PCR protocol was developed to increase sensitivity and specificity of the method. Primers were located on different exons to allow distinguishing between amplified spliced transcripts and contaminating DNA on the basis of their different product sizes (Figure 1). The annealing temperature of the first PCR was chosen below that of the nested PCR to minimize binding of the outer primers to amplification products in the nested PCR. Various annealing temperatures were systematically tested to achieve maximal sensitivity and specificity. First-strand cDNA was synthesized from 1 µg of RNA in a total volume of 20 µL with a reverse transcription kit (Life Technologies Inc) according to the recommendations of the manufacturer and using the outer 3‘PCR-primer 1245rev as the specific primer.

Place holder to copy figure label and caption
Figure 1.

Schematic illustration of the α-fetoprotein reverse transcription–polymerase chain reaction (AFP–RT-PCR). Arrows indicate the position of primer sets used for PCR (1.PCR) and nested PCR (2.PCR). bp indicates base pair; hybrid oligo, oligonucleotide used for hybridization.

Graphic Jump Location

First PCR was performed using primers 775for (TGA GTC AGA AGT TTA CCA AAG) and 1245rev (CTT CTT CTC CTT TAT CTT GGC). Five microliters of RT reaction mixture was used to amplify AFP cDNAs in a total reaction volume of 100 µL containing the above primers, 25 pmol/L each; deoxynucleotide triphosphate, 0.2 mmol/L; magnesium chloride, 1.5 mmol/L; Taq DNA polymerase, 2.5 U; and PCR buffer consisting of Tris-hydrochloride, 20 mmol/L, and potassium chloride, 50 mmol/L (PCR Kit; Life Technologies Inc). Thirty-five cycles of amplification were done at 30-second intervals at temperatures of 93°C, 62°C, and 72°C, with a final extension step of 10 minutes at 72°C. A total of 20 µL of this reaction mixture was then transferred to the nested PCR, which was also performed in a total volume of 100 µL with primers 954for (TGC AAA CTG ACC ACG CTG GAA C) and 1228rev (TGG CAT TCA AGA GGG TTT TCA GTC). Forty cycles of amplification were done at 30-second intervals at temperatures of 93°C, 66°C, and 72°C, with a final extension step of 10 minutes. The PCR products were subjected to electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. They were subsequently blotted onto nylon membranes (Hybond N+, Amersham Life Science, Buckinghamshire, England) and hybridized with a chemiluminescence-labeled oligonucleotide probe (ECL Detection System, Amersham Life Science) comprising nucleotides 993 to 1024 of the AFP cDNA sequence (CAT GCA GAA AAT GAT GAA AAA CCT GAA GGT CT) as recommended by the supplier. Negative (water) and positive controls (HepG2 RNA) were included in all PCRs to exclude contamination and confirm amplification. Quality of RNA and RT of all analyzed samples was confirmed by amplification of glyceraldehyde phosphate dehydrogenase transcripts as described previously.29

PATIENTS

Informed written consent was obtained from all patients. The study protocol was approved by the ethics committee of the University of Heidelberg, Heidelberg, Germany.

Twenty-four patients who underwent surgery for HCC at the Surgical Department, University of Heidelberg, were included in this study (21 men and 3 women; mean age, 54 years) from September 1996 to February 1998. Bone marrow puncture from the iliac crests of both sides was performed under sterile conditions directly after induction of anesthesia. Three blood samples were obtained from each patient through a central venous catheter, the first after induction of anesthesia, the second after resection of the tumor, and the third 24 hours after the operation. Tumor tissue was obtained directly after resection and shock-frozen in liquid nitrogen. Tumor stage and grading was classified according to the fifth edition of the TNM classification of the International Union Against Cancer.30 Serum levels of AFP in peripheral blood were analyzed preoperatively in the routine laboratory workup and measured by commercially available enzyme-linked immunosorbent assay (reference range, <10 µg/L).

Bone marrow puncture was additionally performed on 21 patients with benign diseases (15 men and 6 women; mean age, 49 years). In the majority of these patients, malignant disease had been suspected preoperatively; however, postoperative histological assessment ruled out malignancy. Six of the control patients had benign liver tumors, and the other 15 had other benign tumors of the gastrointestinal tract or chronic inflammations previously suspected to be malignant. Central venous blood samples were also obtained from 13 patients with benign liver diseases as described above for the patients with HCC. Peripheral blood samples were additionally taken from 20 healthy volunteers.

STATISTICAL ANALYSIS

Statistical computations were done using the software packages S-PLUS version 3.4 (MathSoft, Inc, Cambridge, Mass) and StatXact3 for Windows (Sytel Software Corp, Cambridge, Mass). Results were defined as statistically significant when the respective test was P≤.05.

Stage vs RT-PCR Results

The Fisher exact test and Kendall tau test were used to analyze the correlation between tumor stage and detection of AFP mRNA by RT-PCR in bone marrow and blood samples.

RT-PCR Results in Blood Samples vs in Bone Marrow Samples

Odds ratios were calculated and Mantel-Haenszel and Fisher exact tests were performed to analyze the correlation between detection of AFP mRNA in bone marrow and blood samples.

Serum AFP in Peripheral Blood vs RT-PCR Results

Wilcoxon Mann-Whitney, Fisher exact, and Kendall tau tests were used to analyze the correlation between elevated serum AFP levels in peripheral blood and detection of AFP gene products by RT-PCR in bone marrow or blood samples.

SENSITIVITY AND SPECIFICITY OF AFP–RT-PCR

In cell spiking experiments, a 275–base pair PCR product was amplified from samples enriched with HepG2 cells in a dilution of up to 50 cells in an overall volume of 10 mL, thereby amounting to a sensitivity of about 5 cells in 1 mL of blood (approximately corresponding to 5 cells in 1 × 107 peripheral mononuclear cells) (Figure 2). The above PCR fragments were shown by Southern blotting to hybridize to an oligonucleotide probe specific for AFP, thus confirming it as corresponding to an AFP cDNA fragment.

Place holder to copy figure label and caption
Figure 2.

Amplification products of α-fetoprotein–expressing HepG2 cells in serial dilutions (0, 5, 50, 500, 5000 cells) in an overall volume of 10 mL of blood (approximately 108 mononuclear cells) of healthy donors. M indicates molecular weight marker; W, water (negative) control.

Graphic Jump Location

All bone marrow samples from 21 patients without malignant disease and all peripheral blood samples of the 20 healthy volunteers consistently tested negative for AFP expression. Tumor tissue from patients with HCC all tested positive for AFP expression, as expectedly did all normal liver tissue samples. A considerable number of control patients with benign liver diseases, however, showed positive RT-PCR results for AFP mRNA in central venous blood samples (Table 1).

Table Graphic Jump LocationTable 1. Detection Rates of AFP mRNA in Patients With Hepatocellular Carcinoma and Benign Liver Diseases*
PATIENT STUDY
RT-PCR Results and Serum AFP Levels

Four (17%) of the 24 patients with HCC demonstrated AFP-expressing cells in their bone marrow (Figure 3). All 4 of these patients had advanced disease (TNM stage 3 or 4) (Table 2); 2 patients had surgical explorations without resection as the tumor was macroscopically too advanced, 1 patient already had a recurrence of a primarily resected HCC and had to undergo transplantation, and 1 patient had a tumor larger than 2 cm with vascular infiltration. However, not all patients with advanced disease showed positive RT-PCR results and correlation of advanced tumor stage (stage 3 or higher) to AFP expression in bone marrow samples did not reach significance (P = .07). In central venous blood samples, AFP-expressing cells were found in 7 patients preoperatively (29%), in 11 patients intraoperatively (46%), and in 10 patients 24 hours postoperatively (43%) (Table 1). There was no statistically significant difference between the detection of AFP mRNA in central venous blood samples in patients with HCC or in patients with benign liver diseases nor was there any statistically significant association between the detection of AFP mRNA in bone marrow or blood samples, although all patients with AFP-expressing cells in their bone marrow at least had 1 positive blood sample. Serum AFP levels were elevated in all patients with detection of AFP mRNA in their bone marrow; however, many patients with elevated serum AFP levels did not show AFP-expressing cells in bone marrow, therefore no statistically significant correlation could be found between elevated serum AFP levels and positive AFP–RT-PCR results in bone marrow samples. Two of the 4 patients with positive bone marrow results had antibodies against hepatitis B or C, and the other 2 patients did not show any evidence for viral hepatitis (Table 2).

Place holder to copy figure label and caption
Figure 3.

Reverse transcription–polymerase chain reaction (RT-PCR) with Southern blot hybridization of PCR products with an α-fetoprotein (AFP)–specific oligonucleotide. M indicates molecular weight marker; W, water (negative) control; lane 1, negative control cytokeratin-20 amplification product, 290 base pairs (bp) (HT-29 cells); lanes 2-8, AFP amplification products, 275 bp. Lanes 2 to 6 correspond to bone marrow samples of patients 3, 17, 20, 21, and 24; lanes 7 and 8 are positive controls (HepG2 cells).

Graphic Jump Location
Table Graphic Jump LocationTable 2. Clinical Characteristics and AFP mRNA Detection Results in Patients With Hepatocellular Carcinoma*
FOLLOW-UP

Patient 3, who had undergone transplantation because of a recurrence of HCC after R0-resection (microscopically tumor-free resection margins), developed a recurrence in the transplanted liver 6 months later and had to undergo liver resection again. One of the other 2 patients with AFP-expressing cells in their bone marrow samples who did not have resection died of his metastatic tumor (patient 17), and the other is still alive with hepatic progression of the malignant disease (patient 20). Patient 21 is clinically still without recurrence; however, the follow-up is only 4 months.

Two of the 20 patients with HCC and negative RT-PCR results in their bone marrow died perioperatively (both due to septic complications after liver transplantation), and 4 patients died of progression of their malignant disease. Excluding the 2 patients who died perioperatively, the median follow-up is 11 months (range, 4-20 months).

Patients with HCC often develop recurrences, which are probably caused by preoperatively already existing micrometastases. Therefore, preoperative screening for occult tumor cells is important to adequately select patients for therapeutic regimens. Previous studies have indicated a prognostic relevance for the detection of micrometastasis in bone marrow samples in patients with colorectal and other gastrointestinal cancers.2026 Equivocal results have questioned whether this also applies to tumor cell detection in peripheral blood samples of affected cancer patients. Especially in HCC, conflicting results regarding specificity of RT-PCR–based detection systems in blood samples have been published.1319 The major problem is finding an adequate specific marker, which should obviously not be expressed in normal peripheral blood cells. False-positive results may originate from "illegitimate" transcription, as shown for the albumin gene, or from pseudogenes, as shown for cytokeratin 18 and 19, or may simply be caused by contamination due to the high sensitivity of the method.7,31,32

We have developed a sensitive and specific RT-PCR method for the detection of AFP-expressing cells in bone marrow samples. When testing preoperative, intraoperative, or postoperative central venous blood samples from patients operated on for benign liver disease and HCC we found positive RT-PCR results in a comparable percentage in both groups. This is in accordance with Lemoine and colleagues,19 who also investigated AFP expression by RT-PCR in intraoperative blood samples and showed comparable detection rates in intraoperative blood samples of patients with HCC and patients without malignant disease (predominantly patients with liver cirrhosis). The high detection rate in patients with benign and malignant diseases in our study may be explained by the position of the central line, which is routinely placed in or just proximal to the right atrium where blood that has just passed through the liver and drained into the inferior vena cava merges with blood from the upper body. Normal liver cells expressing AFP are continuously shed into the hepatic veins and either undergo apoptosis or are filtered out of the blood system when passing capillary beds. Cell shedding may be enhanced by surgical manipulation and inflammatory conditions within the hepatic parenchyma. Central venous blood in our study was drawn before it passed through any capillary bed only a short distance after leaving the liver, therefore possibly accounting for the high rate of detection. This may also be the reason for conflicting results of detection of AFP-expressing cells in blood samples in the literature as the conditions of blood sampling may not have been comparable. Unfortunately, the exact method of obtaining blood is not specified in most articles. It is necessary to further investigate whether the detection of AFP mRNA in peripheral blood samples that have passed through at least one capillary bed is significantly different from the detection in central venous blood samples. In a subgroup of 8 patients, who had all tested positive for AFP mRNA in intraoperative central venous blood samples, only 6 patients showed AFP expression in intraoperative arterial blood samples, thereby suggesting a difference in the detection rate before and after passage through a capillary bed. Preoperative and postoperative blood samples in our study equally revealed positive RT-PCR results in both groups, which also supports the notion that these AFP-expressing cells in the central venous blood are hepatocytes that are later filtered out of the blood or undergo apoptosis. The former concept is further substantiated by the fact that all 20 healthy controls consistently tested negative for AFP expression in peripheral blood samples, thereby making "illegitimate transcription" as cause for the detection of AFP-expressing cells in central venous blood samples in patients with benign diseases very unlikely. In our study, the detection of AFP mRNA was even higher in intraoperative central venous blood samples of patients with benign liver diseases compared with patients with HCC; however, this is easily explicable by a dilution effect. The latter group lost considerably more blood, which was intraoperatively replaced by blood transfusions. Similar results have been reported from other studies analyzing perioperative tumor cell dissemination where a blood loss of more than 1 L was statistically calculated as cutoff point for reliable intraoperative tumor cell detection.31

On the other hand, we did not detect AFP-expressing cells in bone marrow samples of 21 patients with benign diseases, but did in 4 of 24 patients with HCC. Correlation of AFP expression in bone marrow samples to tumor stage was not statistically significant, probably due to the limited number of tested patients. The prognostic relevance of these data has to be further evaluated by clinical follow-up. Interestingly, 1 of the 4 AFP-positive patients soon developed a cancer recurrence in the transplanted new liver. The other 3 patients all had advanced disease, and no patient below tumor stage 3 showed AFP-expressing cells in bone marrow. It is certainly premature to derive any therapeutic consequences from these results. However, if detection of tumor cells in bone marrow samples is associated with an increase in the recurrence rate, preoperative screening before liver transplantation for HCC seems warranted.

We could also not demonstrate any correlation between elevated AFP serum levels and micrometastasis in bone marrow or blood samples. This is in contrast to the results of Louha et al,15 who found a strong correlation between the presence of circulating AFP-positive cells in peripheral blood samples of patients with HCC and serum AFP concentrations. Interestingly, we did not detect any bone marrow micrometastasis in patients with normal AFP levels. The results of Lemoine et al,19 however, are in accordance with ours as they too were not able to show any correlation between micrometastatic disease in blood samples and elevated AFP serum levels.

In conclusion, we have developed a sensitive, specific test to detect AFP-expressing cells in bone marrow samples. Bone marrow micrometastases were found in 4 of 24 patients with HCC, whereas none of the 21 controls showed positive PCR signals. Potential implications for therapeutic decisions have to be evaluated in further studies and in the clinical follow-up of the tested patients. We believe this assay should be considered in the preoperative workup before liver transplantation to adequately select suitable patients. The AFP–RT-PCR method, however, cannot be recommended for the detection of tumor cell dissemination in blood samples obtained through a central venous catheter from patients with HCC since a large number of control patients with benign liver diseases equally demonstrated AFP-expressing cells. These cells are probably hepatocytes that are regularly shed into the central venous blood and then undergo apoptosis or are filtered out of the blood circulation.

Corresponding author: Peter Kienle, MD, Division of Molecular Diagnostics and Therapy, Department of Surgery, University of Heidelberg, INF 110, D-69120 Heidelberg, Germany (e-mail: p21@ix.urz.uni-heidelberg.de).

Adachi  EMaeda  TMatsumata  T  et al.  Risk factors for intrahepatic recurrence in human small hepatocellular carcinoma. Gastroenterology. 1995;108768- 775
Link to Article
Bismuth  HChiche  LAdam  RCastaing  DDiamond  DDennison  A Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients. Ann Surg. 1993;218145- 151
Link to Article
Farmer  DGRosove  MHShaked  ABusuttil  RW Current treatment modalities for hepatocellular carcinoma. Ann Surg. 1994;219236- 247
Link to Article
Lehnert  TOtto  GHerfarth  CH Therapeutic modalities and prognostic factors for primary and secondary liver tumors. World J Surg. 1993;19252- 263
Link to Article
Ringe  BPichlmayr  RWittekind  CTusch  G Surgical treatment of hepatocellular carcinoma: experience with liver resection and transplantation in 198 patients. World J Surg. 1991;15270- 285
Link to Article
Johnson  PWMBurchill  SASelby  PJ The molecular detection of circulating tumor cells. Br J Cancer. 1995;72268- 276
Link to Article
Neumaier  MGerhard  MWagener  C Diagnosis of micrometastasis by amplification of tissue-specific genes. Gene. 1995;15943- 47
Link to Article
Ghossein  RARosai  J Polymerase chain reaction in the detection of micrometastasis and circulating tumor cells. Cancer. 1996;7810- 16
Link to Article
Carr  BIKar  S An assay for hepatoma micrometastasis: albumin expression in peripheral blood from patients with advanced hepatocellular carcinoma (HCC) [abstract]. Hepatology. 1991;14108A
Hillaire  SBarbu  VBoucher  EMoukthar  MPoupon  R Albumin messenger RNA as a marker of circulating hepatocytes in hepatocellular carcinoma. Gastroenterology. 1994;106239- 242
Chou  HCSheu  JCHuang  GTWang  JTChen  DS Albumin messenger RNA is not specific for circulating hepatoma cells. Gastroenterology. 1994;107630- 631
Niwa  YMatsumara  MShiratori  Y  et al.  Quantitation of α-fetoprotein and albumin messenger RNAs in human hepatocellular carcinoma. Hepatology. 1996;232384- 2392
Funaki  NOTanaka  JSeto  S-IKasamatsu  TKaido  TImamura  M Hematogenous spreading of hepatocellular carcinoma cells: possible participation in recurrence in the liver. Hepatology. 1997;25564- 568
Link to Article
Komeda  TFukuda  YSando  T  et al.  Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood. Cancer. 1995;752214- 2219
Link to Article
Louha  MPoussin  KGanne  N  et al.  Spontaneous and iatrogenic spreading of liver-derived cells into peripheral blood of patients with primary liver cancer. Hepatology. 1997;26998- 1005
Link to Article
Matsumara  MNiwa  YKato  N  et al.  Detection of alpha fetoprotein mRNA, an indicator of hematogenous spreading of hepatocellular carcinoma in the circulation. Hepatology. 1994;201418- 1425
Link to Article
Wong  IH-NLeung  THo  SLau  WYChan  MJohnson  PJ Semiquantitation of circulating hepatocellular carcinoma cells by reverse transcriptase polymerase chain reaction. Br J Cancer. 1997;76628- 633
Link to Article
Jiang  SYShyu  RYHuang  MF  et al.  Detection of alphafetoprotein-expressing cells in the blood of patients with hepatoma and hepatitis. Br J Cancer. 1997;75928- 933
Link to Article
Lemoine  ALe Bricon  TSalvucci  M  et al.  Prospective evaluation of circulating hepatocytes by alpha-fetoprotein mRNA in humans during liver surgery. Ann Surg. 1997;22643- 50
Link to Article
Diel  IJKaufmann  MGörner  RCosta  SDKaul  SBastert  G Detection of tumor cells in bone marrow of patients with primary breast cancer: a prognostic factor for distant metastasis. J Clin Oncol. 1992;101534- 1539
Gerhard  MJuhl  HKalthoff  HSchreiber  HWagener  CNeumaier  M Specific detection of carcinoembryonic antigen–expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol. 1994;12725- 729
Lindemann  FSchlimok  GDischedt  PWitte  JRiethmüller  G Prognostic significance of micrometastatic tumor cells in bone marrow of colorectal cancer patients. Lancet. 1992;340685- 689
Link to Article
Schlimok  GRiethmüller  C Detection, characterization and tumorigenicity of disseminated tumor cells in human bone marrow. Semin Cancer Biol. 1990;1207- 215
Pantel  KIzbicki  JRPasslick  B  et al.  Frequency and prognostic significance of isolated tumor cells in bone marrow of patients with non–small-cell lung cancer without overt metastases. Lancet. 1996;347649- 653
Link to Article
Soeth  ERöder  CJuhl  HKrüger  UKremer  BKalthoff  H The detection of disseminated tumor cells in bone marrow from colorectal cancer patients by a cytokeratin-20–specific nested reverse transcriptase-polymerase chain reaction is related to the stage of disease. Int J Cancer. 1996;69278- 282
Link to Article
Soeth  EVogel  IRöder  C  et al.  Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR. Cancer Res. 1997;573106- 3110
Knowles  BBHowe  CCAden  DP Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980;209497- 499
Link to Article
Gibbs  PEZielinski  RBoyd  CDugaiczyk  A Structure, polymorphism and novel repeated DNA elements revealed by a complete sequence of the human α-fetoprotein gene. Biochemistry. 1987;261332- 1343
Link to Article
Hsu  EMcNicol  PGuijon  FParaskevas  M Quantitation of HPV-16 E6-E7 transcription in cervical intraepithelial neoplasia by reverse transcriptase-polymerase chain reaction. Int J Cancer. 1993;55397- 401
Link to Article
Sobin  LHWittekind  CH UICC: TNM Classification of Malignant Tumors. 5th ed. London, England John Wiley & Sons Inc Publications1997;
Weitz  JKienle  PLacroix  J  et al.  Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res. 1998;4343- 348
Chelly  JConcordet  JPKaplan  JCKahn  A Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci U S A. 1989;862617- 2621
Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Schematic illustration of the α-fetoprotein reverse transcription–polymerase chain reaction (AFP–RT-PCR). Arrows indicate the position of primer sets used for PCR (1.PCR) and nested PCR (2.PCR). bp indicates base pair; hybrid oligo, oligonucleotide used for hybridization.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Amplification products of α-fetoprotein–expressing HepG2 cells in serial dilutions (0, 5, 50, 500, 5000 cells) in an overall volume of 10 mL of blood (approximately 108 mononuclear cells) of healthy donors. M indicates molecular weight marker; W, water (negative) control.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

Reverse transcription–polymerase chain reaction (RT-PCR) with Southern blot hybridization of PCR products with an α-fetoprotein (AFP)–specific oligonucleotide. M indicates molecular weight marker; W, water (negative) control; lane 1, negative control cytokeratin-20 amplification product, 290 base pairs (bp) (HT-29 cells); lanes 2-8, AFP amplification products, 275 bp. Lanes 2 to 6 correspond to bone marrow samples of patients 3, 17, 20, 21, and 24; lanes 7 and 8 are positive controls (HepG2 cells).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Detection Rates of AFP mRNA in Patients With Hepatocellular Carcinoma and Benign Liver Diseases*
Table Graphic Jump LocationTable 2. Clinical Characteristics and AFP mRNA Detection Results in Patients With Hepatocellular Carcinoma*

References

Adachi  EMaeda  TMatsumata  T  et al.  Risk factors for intrahepatic recurrence in human small hepatocellular carcinoma. Gastroenterology. 1995;108768- 775
Link to Article
Bismuth  HChiche  LAdam  RCastaing  DDiamond  DDennison  A Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients. Ann Surg. 1993;218145- 151
Link to Article
Farmer  DGRosove  MHShaked  ABusuttil  RW Current treatment modalities for hepatocellular carcinoma. Ann Surg. 1994;219236- 247
Link to Article
Lehnert  TOtto  GHerfarth  CH Therapeutic modalities and prognostic factors for primary and secondary liver tumors. World J Surg. 1993;19252- 263
Link to Article
Ringe  BPichlmayr  RWittekind  CTusch  G Surgical treatment of hepatocellular carcinoma: experience with liver resection and transplantation in 198 patients. World J Surg. 1991;15270- 285
Link to Article
Johnson  PWMBurchill  SASelby  PJ The molecular detection of circulating tumor cells. Br J Cancer. 1995;72268- 276
Link to Article
Neumaier  MGerhard  MWagener  C Diagnosis of micrometastasis by amplification of tissue-specific genes. Gene. 1995;15943- 47
Link to Article
Ghossein  RARosai  J Polymerase chain reaction in the detection of micrometastasis and circulating tumor cells. Cancer. 1996;7810- 16
Link to Article
Carr  BIKar  S An assay for hepatoma micrometastasis: albumin expression in peripheral blood from patients with advanced hepatocellular carcinoma (HCC) [abstract]. Hepatology. 1991;14108A
Hillaire  SBarbu  VBoucher  EMoukthar  MPoupon  R Albumin messenger RNA as a marker of circulating hepatocytes in hepatocellular carcinoma. Gastroenterology. 1994;106239- 242
Chou  HCSheu  JCHuang  GTWang  JTChen  DS Albumin messenger RNA is not specific for circulating hepatoma cells. Gastroenterology. 1994;107630- 631
Niwa  YMatsumara  MShiratori  Y  et al.  Quantitation of α-fetoprotein and albumin messenger RNAs in human hepatocellular carcinoma. Hepatology. 1996;232384- 2392
Funaki  NOTanaka  JSeto  S-IKasamatsu  TKaido  TImamura  M Hematogenous spreading of hepatocellular carcinoma cells: possible participation in recurrence in the liver. Hepatology. 1997;25564- 568
Link to Article
Komeda  TFukuda  YSando  T  et al.  Sensitive detection of circulating hepatocellular carcinoma cells in peripheral venous blood. Cancer. 1995;752214- 2219
Link to Article
Louha  MPoussin  KGanne  N  et al.  Spontaneous and iatrogenic spreading of liver-derived cells into peripheral blood of patients with primary liver cancer. Hepatology. 1997;26998- 1005
Link to Article
Matsumara  MNiwa  YKato  N  et al.  Detection of alpha fetoprotein mRNA, an indicator of hematogenous spreading of hepatocellular carcinoma in the circulation. Hepatology. 1994;201418- 1425
Link to Article
Wong  IH-NLeung  THo  SLau  WYChan  MJohnson  PJ Semiquantitation of circulating hepatocellular carcinoma cells by reverse transcriptase polymerase chain reaction. Br J Cancer. 1997;76628- 633
Link to Article
Jiang  SYShyu  RYHuang  MF  et al.  Detection of alphafetoprotein-expressing cells in the blood of patients with hepatoma and hepatitis. Br J Cancer. 1997;75928- 933
Link to Article
Lemoine  ALe Bricon  TSalvucci  M  et al.  Prospective evaluation of circulating hepatocytes by alpha-fetoprotein mRNA in humans during liver surgery. Ann Surg. 1997;22643- 50
Link to Article
Diel  IJKaufmann  MGörner  RCosta  SDKaul  SBastert  G Detection of tumor cells in bone marrow of patients with primary breast cancer: a prognostic factor for distant metastasis. J Clin Oncol. 1992;101534- 1539
Gerhard  MJuhl  HKalthoff  HSchreiber  HWagener  CNeumaier  M Specific detection of carcinoembryonic antigen–expressing tumor cells in bone marrow aspirates by polymerase chain reaction. J Clin Oncol. 1994;12725- 729
Lindemann  FSchlimok  GDischedt  PWitte  JRiethmüller  G Prognostic significance of micrometastatic tumor cells in bone marrow of colorectal cancer patients. Lancet. 1992;340685- 689
Link to Article
Schlimok  GRiethmüller  C Detection, characterization and tumorigenicity of disseminated tumor cells in human bone marrow. Semin Cancer Biol. 1990;1207- 215
Pantel  KIzbicki  JRPasslick  B  et al.  Frequency and prognostic significance of isolated tumor cells in bone marrow of patients with non–small-cell lung cancer without overt metastases. Lancet. 1996;347649- 653
Link to Article
Soeth  ERöder  CJuhl  HKrüger  UKremer  BKalthoff  H The detection of disseminated tumor cells in bone marrow from colorectal cancer patients by a cytokeratin-20–specific nested reverse transcriptase-polymerase chain reaction is related to the stage of disease. Int J Cancer. 1996;69278- 282
Link to Article
Soeth  EVogel  IRöder  C  et al.  Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR. Cancer Res. 1997;573106- 3110
Knowles  BBHowe  CCAden  DP Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980;209497- 499
Link to Article
Gibbs  PEZielinski  RBoyd  CDugaiczyk  A Structure, polymorphism and novel repeated DNA elements revealed by a complete sequence of the human α-fetoprotein gene. Biochemistry. 1987;261332- 1343
Link to Article
Hsu  EMcNicol  PGuijon  FParaskevas  M Quantitation of HPV-16 E6-E7 transcription in cervical intraepithelial neoplasia by reverse transcriptase-polymerase chain reaction. Int J Cancer. 1993;55397- 401
Link to Article
Sobin  LHWittekind  CH UICC: TNM Classification of Malignant Tumors. 5th ed. London, England John Wiley & Sons Inc Publications1997;
Weitz  JKienle  PLacroix  J  et al.  Dissemination of tumor cells in patients undergoing surgery for colorectal cancer. Clin Cancer Res. 1998;4343- 348
Chelly  JConcordet  JPKaplan  JCKahn  A Illegitimate transcription: transcription of any gene in any cell type. Proc Natl Acad Sci U S A. 1989;862617- 2621
Link to Article

Correspondence

CME
Also Meets CME requirements for:
Browse CME for all U.S. States
Accreditation Information
The American Medical Association is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The AMA designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 CreditTM per course. Physicians should claim only the credit commensurate with the extent of their participation in the activity. Physicians who complete the CME course and score at least 80% correct on the quiz are eligible for AMA PRA Category 1 CreditTM.
Note: You must get at least of the answers correct to pass this quiz.
Your answers have been saved for later.
You have not filled in all the answers to complete this quiz
The following questions were not answered:
Sorry, you have unsuccessfully completed this CME quiz with a score of
The following questions were not answered correctly:
Commitment to Change (optional):
Indicate what change(s) you will implement in your practice, if any, based on this CME course.
Your quiz results:
The filled radio buttons indicate your responses. The preferred responses are highlighted
For CME Course: A Proposed Model for Initial Assessment and Management of Acute Heart Failure Syndromes
Indicate what changes(s) you will implement in your practice, if any, based on this CME course.
Submit a Comment

Multimedia

Some tools below are only available to our subscribers or users with an online account.

Web of Science® Times Cited: 42

Related Content

Customize your page view by dragging & repositioning the boxes below.

Articles Related By Topic
Related Collections
PubMed Articles
JAMAevidence.com

Care at the Close of Life EDUCATION GUIDES
Integrating Palliative Care for Liver Transplant Candidates