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1 Department of Diagnostic Radiology and Organ Imaging, Prince of Wales
Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong SAR.
2 Department of Chemical Pathology, The Chinese University of Hong Kong, Prince
of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
3 Department of Anatomical and Cellular Pathology, The Chinese University of
Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
SAR.
4 Department of Clinical Oncology, The Chinese University of Hong Kong, Prince
of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
5 Department of Surgery, The Chinese University of Hong Kong, Prince of Wales
Hospital, Shatin, New Territories, Hong Kong SAR.
Received November 5, 2003;
accepted after revision February 16, 2004.
Address correspondence to S. C. H. Yu
(simonyu{at}cuhk.edu.hk).
Abstract
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SUBJECTS AND METHODS. In this prospective study of 32 patients with suspected hepatocellular carcinoma who underwent sonographically guided liver biopsy, a peripheral venous blood sample was obtained before and 5 min after the procedure. Biopsy was performed using an 18-gauge biopsy gun. DNA was extracted from the plasma of the blood samples for methylation-specific polymerase chain reaction. Quantitative measures of the plasma tumor DNA were determined with real-time quantitative polymerase chain reaction, and the amount was expressed as a methylation index (%) in plasma.
RESULTS. Nineteen (59.4%) of 32 patients did not have detectable p16 tumor suppressor gene marker (p16M) in plasma before biopsy, and they showed no detectable plasma p16M after biopsy. Thirteen (65%) of 20 patients had p16M identified in the plasma before liver biopsy. Quantitative analysis of the plasma tumor DNA in these 13 patients showed no statistically significant difference in the methylation index before and after biopsy (p = 0.345, Wilcoxon's signed rank test).
CONCLUSION. No evidence exists that percutaneous liver biopsy results in hematogenous dissemination of hepatocellular carcinoma as shown by quantitative analysis of circulating tumor DNA (p16M) using methylation-specific real-time polymerase chain reaction.
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Liver biopsy is an integral and necessary part of the workup in the diagnosis and management of hepatocellular carcinoma. Small risks are associated with such invasive procedures, (e.g., hemorrhage or infection), but with care these problems can be kept to a minimum and should not affect the outcome. However, the possibility of tumor spread is of more concern because it may alter the extent of the disease and the prognosis for the patient.
Tumor spread may occur locally or distantly. Local spread of disease via needle-tract seeding to the subcutaneous tissue, body wall, or lung has been described but is thought to be rare. However, studies in larger series revealed rates in the range of 2.7–5.1% [1, 2]. Local spread usually did not affect survival of the patient [2].
The possibility of distant spread is of greater importance. Hematogenous dissemination of tumor cells as a result of biopsy is a theoretic possibility, and the resulting extrahepatic metastasis would be expected to have a deleterious effect on the patient's prognosis. Although extrahepatic spread as a result of surgical resection for hepatocellular carcinoma has been reported [3–5], little information is available regarding the possibility of distant tumor spread resulting specifically from biopsy of hepatocellular carcinoma lesions.
Our aim was to find out whether percutaneous biopsy of hepatocellular carcinoma causes significant dissemination of tumor cells into the circulation, as indicated by a postbiopsy rise in circulating tumor DNA, which is represented by hypermethylation of the p16 tumor suppressor gene (p16M).
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DNA Extraction and Methylation-Specific Polymerase Chain Reaction
Details of the laboratory technique have been previously described
[7,
8], and only a brief summary is
given here. From the blood specimen, the plasma was separated by
centrifugation. DNA was extracted from the plasma using a QIAamp blood kit
(Qiagen) according to the manufacturer's protocol for methylation-specific
polymerase chain reaction. The DNA was then treated with bisulfite using a
Cp-Genome DNA modification kit (Oncor) following the manufacturer's
recommendation. Methylated cytosine will remain unchanged, whereas
unmethylated cytosine residue will be converted to uracil, allowing
differentiation between methylated and unmethylated sequences. The modified
DNA was then amplified, using a GeneAmp DNA amplification kit with AmpliTaq
Gold (Perkin-Elmer), into a polymerase. The polymerase chain reaction products
were analyzed by agarose gel electrophoresis and ethidium bromide staining.
The identity of the methylated version of the p16 gene was confirmed by
nonradioactive Southern blot analysis.
Quantitative measures of the plasma tumor DNA were determined with real-time quantitative polymerase chain reaction, which was based on continuous monitoring of fluorogenic polymerase chain reaction. The quantity of methylated p16 was compared to the total amount of both methylated and unmethylated p16 and expressed as a methylation index (%).
Statistical Analysis
The amounts of plasma tumor marker before and after biopsy were compared
using the Wilcoxon's signed rank test.
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The presence of p16M was detected in the tumor tissue specimen of 20 (62.5%) of 32 patients, and p16M was also detected in the plasma of 13 (65%) of these 20 patients. In the plasma of those patients in whom p16M did not occur in the tumor tissue, p16M was always absent (Table 1). For the 19 patients in whom p16M was absent in the plasma before biopsy, p16M findings were also negative in the plasma after biopsy and therefore could not be used as a marker for detection of postbiopsy hematogenous dissemination. In Table 2, we analyzed the relationship between tumor size and the presence of p16M in plasma and found that larger tumor size is probably associated with the presence of p16M in plasma. In the 13 patients in whom plasma p16M findings were positive, quantitative analysis of the plasma p16M in terms of the methylation index before and after biopsy was performed. No statistically significant difference was found in the methylation index values before and after biopsy (Table 3).
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The use of 
-fetoprotein messenger RNA as detected by reverse
transcriptase polymerase chain reaction as a marker of tumor content has been
previously described [9,
10]. By using a quantitative
analysis technique, the quantity of circulating tumor cells at different time
spots can be determined. The use of methylation-specific polymerase chain
reaction for p16M is a more recent development presenting us with a novel and
extremely sensitive method for detection of disseminated tumor DNA
[7]. The p16 tumor suppressor
gene is located on chromosome 9p21, and it is one of the most frequently
altered genes observed in various human neoplasms, including hepatocellular
carcinoma [11,
12]. Inactivation of the p16
gene is frequently found in hepatocellular carcinoma and is believed to be an
important factor in the pathogenesis of hepatocellular carcinoma. Of the
various mechanisms of p16 gene inactivation, hypermethylation (aberrant
methylation) in the promoter region of the gene is thought to be the main
cause leading to development of hepatocellular carcinoma
[13,
14].
Hypermethylation of the p16 gene is detectable in a high proportion of patients with hepatocellular carcinoma. Seventy-three percent of patients with hepatocellular carcinoma show aberrant methylation in the DNA of tumor tissue. DNA containing methylated p16 sequences in the circulation may be detected in 81% of patients with such changes using methylation-specific polymerase chain reaction, and the techniques for quantitative analysis of circulation DNA in plasma is now available [7, 8]. The ability to perform a quantitative analysis of tumor DNA allows quantification of circulating tumor DNA at those particular time spots.
Compared to the hypervascularity of hepatocellular carcinoma and liver parenchyma, soft-tissue structures along the biopsy tract are definitely hypovascular. It is believed that postbiopsy needle tract seeding of tumor cells will be unlikely to lead to immediate elevation of circulating DNA level unless the needle tract passes through a vessel of significant size in the body wall, in which case bleeding through the biopsy tract or evidence of intraperitoneal bleeding would be anticipated. Because bleeding did not occur in our 32 patients, it is reasonable to believe that changes in the plasma DNA level are reliable indicators of hematogenous dissemination and are not affected by needle tract tumor seeding.
In this study, biopsies were performed by an experienced interventional radiologist with a single core of tumor tissue taken and minimal trauma inflicted to the liver. To our knowledge, the influence of biopsy technique and the number of needle passes on the potential risk of hematogenous tumor dissemination is yet to be studied. The size of the tumors biopsied in this study was relatively large, with a median of 9.6 cm and an average ± SD of 9.8 ± 5.3 cm, and large tumor size is usually associated with tumor hypervascularity. Therefore, hypervascularity does not seem to contribute to postbiopsy hematogenous dissemination of tumor cells.
Plasma DNA is rapidly turned over, with a mean half-life of 16.3 min [15]; therefore, the level of plasma DNA at any time spot represents an almost real-time account of DNA quantity as an end result of the balance between the DNA production and clearance rates. A constant DNA level implies the production rate is equivalent to the clearance rate. A rise in DNA level signifies an increase in DNA production rate that outmatches the clearance rate and is thus sensitive and useful for monitoring iatrogenic events. The rapid clearance of plasma DNA makes the investigative approach described in our study less susceptible to false-positive results caused by the persistence of tumor DNA produced by other iatrogenic events, which is an advantage when compared to measurement of tumor cell level, which takes 2–4 weeks to clear up [4]. In considering the timing of the second blood sampling, we have balanced the two mutually counteracting factors of tumor DNA production and clearance. The reason for selecting 5 min as the time point for the collection of the postbiopsy blood sample was to ensure enough time for any tumor cells disseminated from the tumors to travel from the liver to the peripheral venous circulation at the antecubital fossa and to allow time for the tumor DNA to accumulate to a higher concentration. On the other hand, because of the rapid DNA clearance rate, we believe that circulating tumor DNA was being continuously eliminated from the circulation once it was disseminated from the tumors. Moreover, we anticipated that thrombosis and hemostasis at the intratumoral biopsy wounds would preclude continuous shedding of tumor DNA into the circulation. Because we did not have a reason to believe that the tumor DNA production rate after 5 min would outmatch the clearance rate, we were convinced that 5 min would be the time spot at which the concentration of circulating tumor DNA would most likely be maximal.
We think that peripheral blood sampling at the antecubital fossa is adequate for our study and that it is not necessary to collect the blood sample in the inferior vena cava. The liver, spleen, and kidneys are the organs that contribute to the clearance of circulating DNA, not the lungs [16–18]. The accumulated circulating DNA level in the blood collected from the inferior vena cava would be the same as that collected from the antecubital vein.
In conclusion, no evidence exists that percutaneous liver biopsy causes hematogenous dissemination of hepatocellular carcinoma, as shown by quantitative analysis of circulating tumor DNA (p16M) using methylation-specific real-time polymerase chain reaction.
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1 cm) hepatic lesions.
Radiology2001; 218:195
-199
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