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Detection of liver cells in peripheral blood of patients with advanced-stage hepatocellular carcinoma
Detection of Liver Cells in Peripheral Blood of Patients With Advanced-Stage Hepatocellular Carcinoma S I D D H A R T H A K A R AND B R I A N I. C A R R
A large percentage of patients w i t h advanced-stage hepatocellular c a r c i n o m a (HCC) h a v e a recurrence of t u m o r in the liver or lung after primary resection and e v e n after orthotopic liver transplantation. One reason for this m a y be the presence of small n u m b e r s of tumor cells circulating in the blood before surgery or the liberation of tumor cells into circulation during surgical manipulation. We tested this h y p o t h e s i s by m e a s u r i n g messenger RNA (mRNA) for h u m a n albumin gene as a liver cell marker with the highly sensitive reverse transcriptase p o l y m e r a s e chain reaction (RT-PCR) technique. Albumin mRNA w a s not found in peripheral blood from normal h u m a n s (0 of 6), from patients with liver cirrhosis (0 of 10), from other tumors metastatic to liver (0 of 10), or during liver transplant surgery for cirrhosis (0 of 10). In patients with advanced-stage HCC (TNM stages III and IV), albumin m R N A was detected (16 of 17) in peripheral blood. After liver transplantation in the HCC patients, the level of m R N A decreased b e l o w the detectable limit (0 of 9). Three of these patients again had detectable m R N A levels w h e n t h e y h a d recurrence of HCC after liver transplantation. Patients with stage I HCC did not h a v e detectable expression. These results suggest that circulating tumor cells are present in patients with advanced-stage HCC, w h i c h m a y be o n e of the reasons w h y these patients h a v e a h i g h incidence of tumor recurrence after apparently definitive surgical resection and e v e n after liver transplantation. (HEPAT O L O G Y 1995;21:403.407.)
Hepatocellular carcinoma (HCC) is a common malignancy in m a n y parts of the world and is estimated to be responsible for 250,000 deaths worldwide every year. Untreated patients with advanced-stage hepatocellular carcinomas have a mean survival time of less t h a n
4 months. Resection of small tumors or limited-stage disease has been associated with prolonged survival. 1~3 However, patients presenting with advanced stages of the disease (TNM stages III and IV) have high tumor recurrence rates after resection 4 and after orthotopic liver transplantation. ~ Reasons for the high recurrence rates after apparently curative resection surgery include undetected tumor in other parts of the liver, positive resection margins, tumor invasion of the hepatic vasculature, and microscopic metastasis. 4'5 However, the high recurrence rate after liver transplantation requires a different explanation. In view of the known predisposition of HCC to invade the hepatic vessels leading to the systemic circulation, we considered the possibility t h a t HCC may be a micrometastatic disease. The hematogenous spread of cancer cells is believed to be a mechanism of metastasis. 6 However, the detection of circulating tumor cells is rarely successful, 7 probably because of limited sensitivity of existing techniques. Recently, the highly sensitive technique of reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect melanoma cells in peripheral blood, s In this study, we used the RT-PCR technique to detect circulating tumor cells in patients with HCC. Messenger RNA (mRNA) expression of h u m a n albumin gene as a liver-specific cell marker was measured in patient's peripheral blood. We reasoned t h a t any cell producing albumin mRNA in the circulation must be liver derived, and we assumed t h a t any circulating liver cells are likely to be shed from a liver tumor. The experiments described here test this hypothesis. Our preliminary data were reported previously. 9 PATIENTS AND METHODS
Abbreviations: HCC, hepatocellular carcinoma; RT-PCR, reverse transcriptase polymerase chain reaction; mRNA, messenger RNA; PMN, peripheral mononuclear cell; PCR, polymerase chain reaction. From the Pittsburgh Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA. Received January 12, 1994; accepted August 31, 1994. This study was supported in part by National Institutes of Health, Bethesda, MD, grant no. CA 57371 (to B.hC.). Address reprint requests to: Brian Carr, MD, FRCP, PhD, Pittsburgh Transplantation Institute, University of Pittsburgh, E1552 BST, Terrace & Lothrop St, Pittsburgh, PA 15260. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2102-002153.00/0
Normal volunteers, patients with HCC, other cancers metastatic to the liver, and cirrhosis, and patients undergoing liver transplantation for cirrhosis were all from the liver transplantation program of the Pittsburgh Transplantation Institute at the University of Pittsburgh Medical Center. Surgical specimens and blood samples were collected from patients following protocol. Cell C u l t u r e C o n d i t i o n s . The human hepatoma cell line Hep3B and the breast cancer cell line MCF7 were used to establish the sensitivity of the method. The cell lines were cultured in minimal essential medium supplemented with 10% fetal bovine serum (Life Technologies, Bethesda, MD). Medium was changed every fourth day, and cells were subcul-
403
Patient Characteristics.
404
KAR AND CARR
HEPATOLOGYFebruary 1995 TABLE 1. D i s e a s e C h a r a c t e r i s t i c s o f P a t i e n t s W i t h H C C
Patients
Cirrhosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
+ + + + + + + + + + + +
No. of Tumors
Size (cm) (Maximum Diameter)
Portal Vein Invasion
Distant Metastasis
1 4 5 2 1 7 4 6 Multiple 1 Multiple Multiple 1 1
3 3 9 5 20 17 6 20 6 3 5 7 4 5 3 16 4
+ + . + + + + + + . + .
--Spine, adrenal -. . Lung Lung Lung, bone Bone, lung -Adrenal, lung Lung . . --
i
Multiple 1
t u r e d after 7 days a n d plated at a dilution of 1:10. Cell n u m bers were counted by a hemocytometer. Peripheral Mononuclear Cell Purification, Peripheral mononuc]ear cells (PMNs) from h u m a n blood were purified from 10 mL of citrated blood from p a t i e n t s with or without HCC on a one-step g r a d i e n t of Ficoll-Paque (Pharmacia, Piscataway, NJ). Briefly, blood was diluted with a n equal volume of phosphate-buffered saline a n d layered on 10 mL of a Ficoll-Paque step gradient. After centrifugation at 400g for 30 minutes, the PMNs were collected from the interface of Ficoll-Paque a n d plasma. The cells were washed with four times the volu m e of phosphate-buffered saline a n d pelleted by low-speed centrifugation. R N A Extraction. Total RNA was extracted from the PMNs and cultured cells u s i n g the RNAzol kit ( C i n n a Biotex, Houston, TX), which is based on the method described by Chomczynski a n d Sacchi. l° RT.PCR. RNA was converted to complementary DNA a n d amplified by h u m a n a l b u m i n gene specific primers following a previously described method. 11 The forward (5'-GCT CAG TAT CTT CAG CAG TGT CC-3') a n d reverse (5'-TGG GTT GTC ATC TTT GTG GTT C-3') polymerase chain reaction (PCR) primers were chosen in exons 3 a n d 4 of the h u m A n a l b u m i n gene to distinguish a n y DNA c o n t a m i n a t i o n of the RNA s a m p l e J 2 Cyclophilin gene expression was m e a s u r e d as a control u s i n g previously described PCR primers. ~3 A 20-#L reaction was prepared with 1 #g of RNA in a PCR buffer c o n t a i n i n g 1.5 mmol/L MgC12, 0.4 pmol/L primers, 200 #tool/ L deoxynucleoside triphosphates, 200 U of m u r i n e l e u k e m i a virus reverse transcriptase, a n d 2 U of T a q - D N A polymerase (Life Technologies, Bethesda, MD). Reverse t r a n s c r i p t i o n was performed at 37°C for 10 m i n u t e s . I m m e d i a t e l y after reverse transcription, PCR was performed i n the same reaction tube for 30 cycles with I m i n u t e of d e n a t u r a t i o n at 94°C, 1 m i n u t e of a n n e a l i n g at 55°C, a n d 1 m i n u t e of extension at 72°C. A 7-minute extension reaction was performed at the end of the PCR cycles. Ten microliters from the PCR reaction was r u n on a 6% polyacrylamide gel t h a t was t h e n stained by e t h i d i u m bromide. DNA Sequencing. DNA sequence was d e t e r m i n e d by the
Hepatitis
B C C C . ----B B C . B C C .
--
Lung .
Time of Recurrence (months)
.
--7 -2 38 12 4 -13 6 --4
dideoxy method u s i n g a kit from USB (Cleveland, OH) a n d the PCR primers as the sequencing primers. RESULTS
N o r m a l v o l u n t e e r s w e r e s e l e c t e d f r o m t h e s t a f f of t h e T r a n s p l a n t a t i o n I n s t i t u t e . O f t h e 10 p a t i e n t s w i t h l i v e r cirrhosis but without HCC, 5 were caused by hepatitis B a n d / o r C, 3 w e r e c a u s e d b y alcohol, a n d 2 w e r e of u n k n o w n cause. This s a m e group was followed d u r i n g l i v e r t r a n s p l a n t a t i o n . N o n H C C t u m o r s m e t a s t a t i c to l i v e r w e r e f r o m c h o l a n g i o c a r c i n o m a (4), colon carcin o m a (3), p a n c r e a t i c c a n c e r (2), a n d g a s t r i c c a n c e r (1). T h e p a t i e n t s w i t h H C C a r e d e s c r i b e d i n T a b l e 1. T h e H e p 3 B cell l i n e w a s e s t a b l i s h e d f r o m a p r i m a r y h u m a n H C C a n d w a s r e p o r t e d to p r o d u c e s e r u m a l b u m i n a n d m a n y m a r k e r s for p r i m a r y H C C . 14 I t w a s a l s o f o u n d to b e t u m o r i g e n i c i n a n a n i m a l m o d e l . T h e
A
Albumin
B
C
D
M
-
FIG. 1. Albumin mRNA detection limit. Different numbers of albumin expressing Hep3B human hepatoma cells were mixed with 1 x 10~ MCF 7 (nonalbumin-producingbreast cancer) cells, and RNA extracted from them were amplified by RT-PCR. The PCR fragments were analyzed on 6% polyacrylamide gel and stained with ethidium bromide. Lanes A, B, C, and D had a mixture of MCF 7 cells and 5,000,500, 50, and 5 Hep3B cells, respectively. Lane M had a molecu]ar weight marker.
HEPATOLOGY Vol. 21, No. 2, 1995
KAR AND CARR
TABLE 2. A l b u m i n m R N A E x p r e s s i o n i n P e r i p h e r a l B l o o d No. of Patients Expressing Albumin mRNA in Circulating Cells Among Total No. of Patients
Patients
0 0 0 0 0 16
Normal Cirrhosis Liver t r a n s p l a n t a t i o n (without HCC) Other tumors metastatic to liver HCC (stage I) HCC (stages III and IV) After liver t r a n s p l a n t a t i o n of HCC (stages III and IV) HCC (recurrence within 6 months)
of 6 of 10 of 10 of 10 of 10 of 17
0 of 9 3 of 3
amount of h u m a n albumin mRNA produced by this cell line was found to be comparable to that produced by primary HCCs (our observation). The sensitivity of the RT-PCR method was determined by mixing HCC cells (Hep3B) together with breast tumor cells (MCF 7), which did not produce albumin. Different numbers of Hep3B cells were added to 1 × 10 s MCF 7 cells. Total RNA was extracted from cells and subjected to RTPCR. The resulting 2 4 9 - b a s e pair amplified fragment was visualized on a 6% polyacrylamide gel after staining with ethidium bromide. The limit of detection was found to be approximately five Hep3B cells in a population of i × 10 ~ MCF 7 cells (Fig. 1). The DNA sequence of 80 bases of the 2 4 9 - b a s e pair amplified fragment was determined to confirm it as a h u m a n albumin sequence. It was found to have a perfect homology to the published sequence for the h u m a n albumin gene from base position 2,381 to base position 2,46012 (data not shown). When normal h u m a n blood was mixed with isolated h u m a n hepatocytes and fractionated in the FicollPaque gradient, the hepatocytes were found to fractionate with the PMNs (our observation). The PMN fractions were isolated from the blood of normal h u m a n volunteers and patients. Total RNA was extracted from
1 |-
2 •
~
these cells, and the RNA was subjected to RT-PCR for h u m a n albumin gene. No freely circulating cell-free albumin mRNA was detected in the peripheral blood of patients with HCC if RT-PCR was performed with the PMN fraction without RNA extraction. Results of detectable albumin mRNA in peripheral blood are summarized in Table 2. No albumin mRNA was detected in normal h u m a n blood (0 of 6), blood of patients with liver cirrhosis (0 of 10), other tumors metastatic to liver, or blood of patients undergoing liver transplantation without any cancer (0 of 10). However, albumin mRNA was detected in the blood of patients (no. 1 to 16 in Table 1) with advanced stages III and IV HCC (16 of 17) (Fig. 2) but not in the blood of patients with minute stage I (a gift from Kurume University, Fukuoka, Japan) HCC (0 of 10) (data not shown). Interestingly, no albumin mRNA could be detected after liver transplantation in the patients with advanced stage HCC (0 of 9) (no. 1, 2, 3, 4, 6, 9, 12, 13, and 16 in Table 1) and (Fig. 2). Three of these patients (no. 6, 9, and 16 in Table 1) again had detectable albumin mRNA when their tumors recurred after liver transplantation (Fig. 3). All of these patients with recurrence had portal vein invasion in the original tumor. The RT-PCR results were quite reproducible when independent samples were obtained from the same patient or when RTPCR was repeated on the same sample. DISCUSSION
H u m a n albumin mRNA could be detected in the peripheral blood of patients with advanced-stage but not early-stage HCC by RT-PCR. This may be caused by the invasion of the hepatic portal vessels, leading to the systemic circulation in the more advanced stages of the disease. A characteristic feature of h u m a n HCC involves tumor invasion of portal vessels, particularly in larger tumors. ~s In the circulation, mRNA was not derived from lysed hepatocytes in the patients with HCC because no signal was detected after RT-PCR when the isolated cells from the peripheral blood was not lysed for RNA extraction. This mRNA was probably
3 B
B A B A BA F]G. 2. Albumin mRNA expression in peripheral blood of patients with stages III and IV HCC before and after liver transplantation. Lanes B and A are samples from before and after liver t r a n s p l a n t a t i o n of four patients (no. 1, 2, 3, and 4). Lane M is a size marker. Cyclophilin expression is used as a control in this figure and in Figs. 2 and 3.
405
4 •
|
•
B AM
C - Cyclophilin -Albumin
406
KAR AND CARR
HEPATOLOGYFebruary 1995
1 •
M
|
BA
R
B
2
•
~1111
3
•
B A R B A R - Cyclophilin - Albumin
also not caused by illegitimate transcription in the blood cells as reported previously 16 because the signal was absent in normal blood and in blood of other patients without primary HCC. The mRNA was most likely derived from malignant hepatocytes from the HCC and not from normal hepatocytes shed from the liver because of the presence of the primary tumor, because patients with other secondary tumors to the liver or patients with liver cirrhosis did not have detectable mRNA. However, the possibility t h a t the albumin mRNA was derived from benign liver cells t h a t were liberated in the circulation as a result of the presence of the primary tumor cannot be strictly ruled out. P r i m a r y tumors may have different characteristics from the metastatic tumors in this regard. No albumin mRNA was found in patients undergoing liver transplantation without any cancer. This suggests t h a t either hepatocytes were not liberated in the circulation because of surgical manipulation, or even if a few cells were liberated, they were lower t h a n our limit of detection. In patients with HCC who underwent liver transplantation, the mRNA signal disappeared from circulating cells after the transplantation, suggesting a limited life span of these cells. Because of the removal of the HCC by transplantation, no new cells could be liberated into the circulation, and as a result, the number of circulating tumor cells decreased below the detectable limit. However, the high tumor recurrence rates even after liver transplantation suggest t h a t the circulating tumor cells did not disappear completely. Reappearance of the circulating tumor cells occurred in all three patients who had clinically detectable recurrence of their HCC. Long-term serial samples were not obtained in this patient series; therefore, we do not yet know if this assay can be used to predict tumor recurrence. This may also be a useful assay to monitor the effectiveness of chemotherapy, surgical resection, or transplantation. After submission of this report, we learned of a simi-
FIG. 3. Albumin mRNA expression in patients with a recurrence of HCC. Three HCC patients before (lane B) and after (lane A) liver transplantation and after recurrence (lane R) are shown. Lanes M and C are marker and control, respectively.
lar study by Hillaire et al. 17 In the report, Hillaire et al. used the same experimental method t h a t was first proposed by us, 9 and they were also able to detect circulating liver cells in patients with HCC. One aspect t h a t is presented in our report t h a t was not studied by Hillaire et al is our analysis of patients with HCC who underwent liver transplantation. We saw a disappearance of the albumin mRNA signal after liver transplantation of patients with HCC. When recurrence of liver tumors was observed in some of these patients, the mRNA became detectable again. This probably constitutes a more direct proof of an association between HCC and detectable albumin mRNA in the peripheral blood of these patients. REFERENCES
1. Zhou X-D, Tang Z-Y, Yu Y-Q, Yang B-H, Lin Z-Y, Lu J-Z, Ma ZC, et al. Long-term survivors after resection for primary liver cancer. Cancer 1989;63:2201-2206. 2. Okuda K. Hepatocellular carcinoma: recent progress. HEPATOLOG¥ 1992; 15:948-963. 3. Ringe B, Pichhnayr R, Wittekind C, Tusch G. Surgical treatment of hepatocellular carcinoma: experience with liver resection and transplantation in 198 patients. World J Surg 1991; 15:270-285. 4. Nagasue N, Uchida M, Makino Y, Takemoto Y, Yamanoi A, Hayashi T, Chang Y-F, et a]. Incidence and factors associated with intrahepatic recurrence following resection of hepatocellular carcinoma. Gastroenterology 1993; 105:488-494. 5. Yokoyama I, Todo S, Iwatsuki S, Starzl TE. Liver transplantation in the treatment of primary liver cancer. Hepatogastroenterology 1990;37:188-193. 6. Tarin D, Price JE, Kettlewell MGW, Souter RG, Vass ACR, Crossley B. Clinicopathological observations on metastases in man studied in patients treated witb peritoneovenous shunts. Br Med J 1984;288:749-751. 7. Moss TJ, Sanders DGJ. Detection of neuroblastoma cells in blood. J Clin Oncol 1990;8:736-740. 8. Smith B, Selby P, Southgate J, Pittman K, Bradley C, Blair GE. Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 1991;338:1227-1229. 9. Carr BI, Kar S. An assay for hepatoma micrometastasis: albumin gene expression in peripheral blood from patients with advanced hepatocellular carcinoma (abstr). HEPATOLOGY1991; 14: A242.
HEPATOLOGYVol. 21, No. 2, 1995 10. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. 11. Mivechi NF, Rossi JJ. Use of polymerase chain reaction to detect the expression of the Mr 70,000 heat shock genes in control or heat shock leukemia cells as correlated to their heat response. Cancer Res 1990;50:2877-2884. 12. Minghetti PP, Ruffner DE, Kuang W-J, Dennison OE, Hawkins JV¢, Beattie WG, Dugaiczyk A. A molecular structure of the human albumin gene is revealed by nucleotide sequence within @1-22 of chromosome 4. J Biol Chem 1986;261:6747-6757. 13. Liu J, Albero MW, Chen C-M, Schreiber SL, Walsh CT. Cloning, expression and purification of human cyclophilin in Escherichia coli and assessment of the catalytic role of cysteines by site-
KAR AND CARR
14. 15. 16. 17.
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directed mutagenesis. Proc Natl Acad Sci USA 1990;87:23042308. Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete major plasma proteins and hepatitis B surface antigen. Science 1980;209:497-499. Ohto M, Kondo F, Ebara M. Pathology, diagnosis and treatment for small liver cancer. In: Tobe T, et al, eds. Primary liver cancer in Japan. Tokyo: Springer-Verlag, 1992:365-373. Chelly J, Concordet JP, Kaplan JC, Khan A. Illegitimate transcription of any gene in any cell type. Prec Natl Acad Sci USA 1986;86:2617-2621. Hillaire S, Barbu V, Boucher E, Moukhter M, Poupon R. Albumin mRNA as a marker of circulating hepatocytes in hepatocellular carcinoma. Gastroenterology 1994; 106:239-242.
A large percentage of patients w i t h advanced-stage hepatocellular c a r c i n o m a (HCC) h a v e a recurrence of t u m o r in the liver or lung after primary resection and e v e n after orthotopic liver transplantation. One reason for this m a y be the presence of small n u m b e r s of tumor cells circulating in the blood before surgery or the liberation of tumor cells into circulation during surgical manipulation. We tested this h y p o t h e s i s by m e a s u r i n g messenger RNA (mRNA) for h u m a n albumin gene as a liver cell marker with the highly sensitive reverse transcriptase p o l y m e r a s e chain reaction (RT-PCR) technique. Albumin mRNA w a s not found in peripheral blood from normal h u m a n s (0 of 6), from patients with liver cirrhosis (0 of 10), from other tumors metastatic to liver (0 of 10), or during liver transplant surgery for cirrhosis (0 of 10). In patients with advanced-stage HCC (TNM stages III and IV), albumin m R N A was detected (16 of 17) in peripheral blood. After liver transplantation in the HCC patients, the level of m R N A decreased b e l o w the detectable limit (0 of 9). Three of these patients again had detectable m R N A levels w h e n t h e y h a d recurrence of HCC after liver transplantation. Patients with stage I HCC did not h a v e detectable expression. These results suggest that circulating tumor cells are present in patients with advanced-stage HCC, w h i c h m a y be o n e of the reasons w h y these patients h a v e a h i g h incidence of tumor recurrence after apparently definitive surgical resection and e v e n after liver transplantation. (HEPAT O L O G Y 1995;21:403.407.)
Hepatocellular carcinoma (HCC) is a common malignancy in m a n y parts of the world and is estimated to be responsible for 250,000 deaths worldwide every year. Untreated patients with advanced-stage hepatocellular carcinomas have a mean survival time of less t h a n
4 months. Resection of small tumors or limited-stage disease has been associated with prolonged survival. 1~3 However, patients presenting with advanced stages of the disease (TNM stages III and IV) have high tumor recurrence rates after resection 4 and after orthotopic liver transplantation. ~ Reasons for the high recurrence rates after apparently curative resection surgery include undetected tumor in other parts of the liver, positive resection margins, tumor invasion of the hepatic vasculature, and microscopic metastasis. 4'5 However, the high recurrence rate after liver transplantation requires a different explanation. In view of the known predisposition of HCC to invade the hepatic vessels leading to the systemic circulation, we considered the possibility t h a t HCC may be a micrometastatic disease. The hematogenous spread of cancer cells is believed to be a mechanism of metastasis. 6 However, the detection of circulating tumor cells is rarely successful, 7 probably because of limited sensitivity of existing techniques. Recently, the highly sensitive technique of reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect melanoma cells in peripheral blood, s In this study, we used the RT-PCR technique to detect circulating tumor cells in patients with HCC. Messenger RNA (mRNA) expression of h u m a n albumin gene as a liver-specific cell marker was measured in patient's peripheral blood. We reasoned t h a t any cell producing albumin mRNA in the circulation must be liver derived, and we assumed t h a t any circulating liver cells are likely to be shed from a liver tumor. The experiments described here test this hypothesis. Our preliminary data were reported previously. 9 PATIENTS AND METHODS
Abbreviations: HCC, hepatocellular carcinoma; RT-PCR, reverse transcriptase polymerase chain reaction; mRNA, messenger RNA; PMN, peripheral mononuclear cell; PCR, polymerase chain reaction. From the Pittsburgh Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA. Received January 12, 1994; accepted August 31, 1994. This study was supported in part by National Institutes of Health, Bethesda, MD, grant no. CA 57371 (to B.hC.). Address reprint requests to: Brian Carr, MD, FRCP, PhD, Pittsburgh Transplantation Institute, University of Pittsburgh, E1552 BST, Terrace & Lothrop St, Pittsburgh, PA 15260. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2102-002153.00/0
Normal volunteers, patients with HCC, other cancers metastatic to the liver, and cirrhosis, and patients undergoing liver transplantation for cirrhosis were all from the liver transplantation program of the Pittsburgh Transplantation Institute at the University of Pittsburgh Medical Center. Surgical specimens and blood samples were collected from patients following protocol. Cell C u l t u r e C o n d i t i o n s . The human hepatoma cell line Hep3B and the breast cancer cell line MCF7 were used to establish the sensitivity of the method. The cell lines were cultured in minimal essential medium supplemented with 10% fetal bovine serum (Life Technologies, Bethesda, MD). Medium was changed every fourth day, and cells were subcul-
403
Patient Characteristics.
404
KAR AND CARR
HEPATOLOGYFebruary 1995 TABLE 1. D i s e a s e C h a r a c t e r i s t i c s o f P a t i e n t s W i t h H C C
Patients
Cirrhosis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
+ + + + + + + + + + + +
No. of Tumors
Size (cm) (Maximum Diameter)
Portal Vein Invasion
Distant Metastasis
1 4 5 2 1 7 4 6 Multiple 1 Multiple Multiple 1 1
3 3 9 5 20 17 6 20 6 3 5 7 4 5 3 16 4
+ + . + + + + + + . + .
--Spine, adrenal -. . Lung Lung Lung, bone Bone, lung -Adrenal, lung Lung . . --
i
Multiple 1
t u r e d after 7 days a n d plated at a dilution of 1:10. Cell n u m bers were counted by a hemocytometer. Peripheral Mononuclear Cell Purification, Peripheral mononuc]ear cells (PMNs) from h u m a n blood were purified from 10 mL of citrated blood from p a t i e n t s with or without HCC on a one-step g r a d i e n t of Ficoll-Paque (Pharmacia, Piscataway, NJ). Briefly, blood was diluted with a n equal volume of phosphate-buffered saline a n d layered on 10 mL of a Ficoll-Paque step gradient. After centrifugation at 400g for 30 minutes, the PMNs were collected from the interface of Ficoll-Paque a n d plasma. The cells were washed with four times the volu m e of phosphate-buffered saline a n d pelleted by low-speed centrifugation. R N A Extraction. Total RNA was extracted from the PMNs and cultured cells u s i n g the RNAzol kit ( C i n n a Biotex, Houston, TX), which is based on the method described by Chomczynski a n d Sacchi. l° RT.PCR. RNA was converted to complementary DNA a n d amplified by h u m a n a l b u m i n gene specific primers following a previously described method. 11 The forward (5'-GCT CAG TAT CTT CAG CAG TGT CC-3') a n d reverse (5'-TGG GTT GTC ATC TTT GTG GTT C-3') polymerase chain reaction (PCR) primers were chosen in exons 3 a n d 4 of the h u m A n a l b u m i n gene to distinguish a n y DNA c o n t a m i n a t i o n of the RNA s a m p l e J 2 Cyclophilin gene expression was m e a s u r e d as a control u s i n g previously described PCR primers. ~3 A 20-#L reaction was prepared with 1 #g of RNA in a PCR buffer c o n t a i n i n g 1.5 mmol/L MgC12, 0.4 pmol/L primers, 200 #tool/ L deoxynucleoside triphosphates, 200 U of m u r i n e l e u k e m i a virus reverse transcriptase, a n d 2 U of T a q - D N A polymerase (Life Technologies, Bethesda, MD). Reverse t r a n s c r i p t i o n was performed at 37°C for 10 m i n u t e s . I m m e d i a t e l y after reverse transcription, PCR was performed i n the same reaction tube for 30 cycles with I m i n u t e of d e n a t u r a t i o n at 94°C, 1 m i n u t e of a n n e a l i n g at 55°C, a n d 1 m i n u t e of extension at 72°C. A 7-minute extension reaction was performed at the end of the PCR cycles. Ten microliters from the PCR reaction was r u n on a 6% polyacrylamide gel t h a t was t h e n stained by e t h i d i u m bromide. DNA Sequencing. DNA sequence was d e t e r m i n e d by the
Hepatitis
B C C C . ----B B C . B C C .
--
Lung .
Time of Recurrence (months)
.
--7 -2 38 12 4 -13 6 --4
dideoxy method u s i n g a kit from USB (Cleveland, OH) a n d the PCR primers as the sequencing primers. RESULTS
N o r m a l v o l u n t e e r s w e r e s e l e c t e d f r o m t h e s t a f f of t h e T r a n s p l a n t a t i o n I n s t i t u t e . O f t h e 10 p a t i e n t s w i t h l i v e r cirrhosis but without HCC, 5 were caused by hepatitis B a n d / o r C, 3 w e r e c a u s e d b y alcohol, a n d 2 w e r e of u n k n o w n cause. This s a m e group was followed d u r i n g l i v e r t r a n s p l a n t a t i o n . N o n H C C t u m o r s m e t a s t a t i c to l i v e r w e r e f r o m c h o l a n g i o c a r c i n o m a (4), colon carcin o m a (3), p a n c r e a t i c c a n c e r (2), a n d g a s t r i c c a n c e r (1). T h e p a t i e n t s w i t h H C C a r e d e s c r i b e d i n T a b l e 1. T h e H e p 3 B cell l i n e w a s e s t a b l i s h e d f r o m a p r i m a r y h u m a n H C C a n d w a s r e p o r t e d to p r o d u c e s e r u m a l b u m i n a n d m a n y m a r k e r s for p r i m a r y H C C . 14 I t w a s a l s o f o u n d to b e t u m o r i g e n i c i n a n a n i m a l m o d e l . T h e
A
Albumin
B
C
D
M
-
FIG. 1. Albumin mRNA detection limit. Different numbers of albumin expressing Hep3B human hepatoma cells were mixed with 1 x 10~ MCF 7 (nonalbumin-producingbreast cancer) cells, and RNA extracted from them were amplified by RT-PCR. The PCR fragments were analyzed on 6% polyacrylamide gel and stained with ethidium bromide. Lanes A, B, C, and D had a mixture of MCF 7 cells and 5,000,500, 50, and 5 Hep3B cells, respectively. Lane M had a molecu]ar weight marker.
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TABLE 2. A l b u m i n m R N A E x p r e s s i o n i n P e r i p h e r a l B l o o d No. of Patients Expressing Albumin mRNA in Circulating Cells Among Total No. of Patients
Patients
0 0 0 0 0 16
Normal Cirrhosis Liver t r a n s p l a n t a t i o n (without HCC) Other tumors metastatic to liver HCC (stage I) HCC (stages III and IV) After liver t r a n s p l a n t a t i o n of HCC (stages III and IV) HCC (recurrence within 6 months)
of 6 of 10 of 10 of 10 of 10 of 17
0 of 9 3 of 3
amount of h u m a n albumin mRNA produced by this cell line was found to be comparable to that produced by primary HCCs (our observation). The sensitivity of the RT-PCR method was determined by mixing HCC cells (Hep3B) together with breast tumor cells (MCF 7), which did not produce albumin. Different numbers of Hep3B cells were added to 1 × 10 s MCF 7 cells. Total RNA was extracted from cells and subjected to RTPCR. The resulting 2 4 9 - b a s e pair amplified fragment was visualized on a 6% polyacrylamide gel after staining with ethidium bromide. The limit of detection was found to be approximately five Hep3B cells in a population of i × 10 ~ MCF 7 cells (Fig. 1). The DNA sequence of 80 bases of the 2 4 9 - b a s e pair amplified fragment was determined to confirm it as a h u m a n albumin sequence. It was found to have a perfect homology to the published sequence for the h u m a n albumin gene from base position 2,381 to base position 2,46012 (data not shown). When normal h u m a n blood was mixed with isolated h u m a n hepatocytes and fractionated in the FicollPaque gradient, the hepatocytes were found to fractionate with the PMNs (our observation). The PMN fractions were isolated from the blood of normal h u m a n volunteers and patients. Total RNA was extracted from
1 |-
2 •
~
these cells, and the RNA was subjected to RT-PCR for h u m a n albumin gene. No freely circulating cell-free albumin mRNA was detected in the peripheral blood of patients with HCC if RT-PCR was performed with the PMN fraction without RNA extraction. Results of detectable albumin mRNA in peripheral blood are summarized in Table 2. No albumin mRNA was detected in normal h u m a n blood (0 of 6), blood of patients with liver cirrhosis (0 of 10), other tumors metastatic to liver, or blood of patients undergoing liver transplantation without any cancer (0 of 10). However, albumin mRNA was detected in the blood of patients (no. 1 to 16 in Table 1) with advanced stages III and IV HCC (16 of 17) (Fig. 2) but not in the blood of patients with minute stage I (a gift from Kurume University, Fukuoka, Japan) HCC (0 of 10) (data not shown). Interestingly, no albumin mRNA could be detected after liver transplantation in the patients with advanced stage HCC (0 of 9) (no. 1, 2, 3, 4, 6, 9, 12, 13, and 16 in Table 1) and (Fig. 2). Three of these patients (no. 6, 9, and 16 in Table 1) again had detectable albumin mRNA when their tumors recurred after liver transplantation (Fig. 3). All of these patients with recurrence had portal vein invasion in the original tumor. The RT-PCR results were quite reproducible when independent samples were obtained from the same patient or when RTPCR was repeated on the same sample. DISCUSSION
H u m a n albumin mRNA could be detected in the peripheral blood of patients with advanced-stage but not early-stage HCC by RT-PCR. This may be caused by the invasion of the hepatic portal vessels, leading to the systemic circulation in the more advanced stages of the disease. A characteristic feature of h u m a n HCC involves tumor invasion of portal vessels, particularly in larger tumors. ~s In the circulation, mRNA was not derived from lysed hepatocytes in the patients with HCC because no signal was detected after RT-PCR when the isolated cells from the peripheral blood was not lysed for RNA extraction. This mRNA was probably
3 B
B A B A BA F]G. 2. Albumin mRNA expression in peripheral blood of patients with stages III and IV HCC before and after liver transplantation. Lanes B and A are samples from before and after liver t r a n s p l a n t a t i o n of four patients (no. 1, 2, 3, and 4). Lane M is a size marker. Cyclophilin expression is used as a control in this figure and in Figs. 2 and 3.
405
4 •
|
•
B AM
C - Cyclophilin -Albumin
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HEPATOLOGYFebruary 1995
1 •
M
|
BA
R
B
2
•
~1111
3
•
B A R B A R - Cyclophilin - Albumin
also not caused by illegitimate transcription in the blood cells as reported previously 16 because the signal was absent in normal blood and in blood of other patients without primary HCC. The mRNA was most likely derived from malignant hepatocytes from the HCC and not from normal hepatocytes shed from the liver because of the presence of the primary tumor, because patients with other secondary tumors to the liver or patients with liver cirrhosis did not have detectable mRNA. However, the possibility t h a t the albumin mRNA was derived from benign liver cells t h a t were liberated in the circulation as a result of the presence of the primary tumor cannot be strictly ruled out. P r i m a r y tumors may have different characteristics from the metastatic tumors in this regard. No albumin mRNA was found in patients undergoing liver transplantation without any cancer. This suggests t h a t either hepatocytes were not liberated in the circulation because of surgical manipulation, or even if a few cells were liberated, they were lower t h a n our limit of detection. In patients with HCC who underwent liver transplantation, the mRNA signal disappeared from circulating cells after the transplantation, suggesting a limited life span of these cells. Because of the removal of the HCC by transplantation, no new cells could be liberated into the circulation, and as a result, the number of circulating tumor cells decreased below the detectable limit. However, the high tumor recurrence rates even after liver transplantation suggest t h a t the circulating tumor cells did not disappear completely. Reappearance of the circulating tumor cells occurred in all three patients who had clinically detectable recurrence of their HCC. Long-term serial samples were not obtained in this patient series; therefore, we do not yet know if this assay can be used to predict tumor recurrence. This may also be a useful assay to monitor the effectiveness of chemotherapy, surgical resection, or transplantation. After submission of this report, we learned of a simi-
FIG. 3. Albumin mRNA expression in patients with a recurrence of HCC. Three HCC patients before (lane B) and after (lane A) liver transplantation and after recurrence (lane R) are shown. Lanes M and C are marker and control, respectively.
lar study by Hillaire et al. 17 In the report, Hillaire et al. used the same experimental method t h a t was first proposed by us, 9 and they were also able to detect circulating liver cells in patients with HCC. One aspect t h a t is presented in our report t h a t was not studied by Hillaire et al is our analysis of patients with HCC who underwent liver transplantation. We saw a disappearance of the albumin mRNA signal after liver transplantation of patients with HCC. When recurrence of liver tumors was observed in some of these patients, the mRNA became detectable again. This probably constitutes a more direct proof of an association between HCC and detectable albumin mRNA in the peripheral blood of these patients. REFERENCES
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