Comparison of Portal Vein Chemotherapy with Hepatic Artery Chemotherapy in the Treatment of Liver Micrometastases Stephen G. Archer, MBBS,Bruce N. Gray, FRACS, PhD, Perth,WesternAustralia
This study was conducted in a rat model of hepatic micrometastases generated by the intraportal injection of a colonic carcinoma cell line bound to polystyrene microspheres. Thirty-two animals received continuous infusions of 5-fluorouracil (5-FU) into either the portal vein or hepatic artery for a period of 7 days. Drug infusions were begun at O, 2, 4, and 6 days after the time of tumor inoculation in four groups of animals, respectively. Subsequent tumor growth in these animals at 1 month was compared with tumor growth in 11 control animals that did not receive chemotherapy. When 5-FU was administered via the portal vein On the same day as tumor inoculation, liver metastases were reduced by approximately 91% (p = 0 . 0 0 4 ) . Portal vein chemotherapy administered 6 days after tumor inoculation, when macroscopic nodules had a mean diameter of 0.33 + 0.03 mm ( S E ) , produced no tumor response (p = 0 . 3 6 ) . Histologic examination of these lesions revealed early invasion outside distended portal venules. In contrast, hepatic artery 5-FU infusions administered at 0, 2, 4, and 6 days after tumor implantation all reduced the subsequent development of hepatic metastases by approximately two thirds of that observed in the untreated group (p = 0 . 0 0 4 ) . We conclude that hepatic artery chemotherapy may have an important complementary role to play as an adjuvant treatment for gastrointestinal cancer.
From the University Department of Surgery, Royal Perth Hospital, Perth, Western Australia. Supported by the Royal Perth Hospital Research Foundation and the Royal Australasian College of Surgeons Research Foundation. Requests for reprints should be addressed to Stephen G. Archer, MBBS, University Department Of Surgery, Royal Perth Hospital, Perth, Western Australia, 6000. Manuscript submitted November 10, 1988, revised April 10, 1989, and accepted April 18, 1989.
n approximately 70% of patients with colorectal canItimecer, the disease appears limited to the bowel at the of surgical resection; in most patients, however, dissemination will occur. The liver is the most common site of recurrence, accounting for up to 30% of treatment failures [1-3]. Some hepatic recurrences may be attributed to the seeding of tumor cells into portal venous blood during and after surgical manipulation of the primary tumor. Others may represent the progression of established metastases that are clinically undetectable at the time of surgery [4]. Adjuvant systemic chemotherapy has been shown to be of little benefit in patients with operable bowel cancer [5]. However, two recent clinical trials have shown a significant survival advantage for patients receiving regional postoperative chemotherapy. The first of these trials, by Taylor et al [6], demonstrated a significant survival advantage for patients with stage B or C colonic carcinoma receiving 7 days of portal vein 5-fluorouracil (5-FU). Subsequently, the Australia and New Zealand Bowel Cancer Trial has confirmed the findings in stage C disease [7]. The survival advantage from portal vein administration of 5-FU primarily stems from a reduction in the incidence of liver metastases. However, not all patients with clinically normal livers at the time of resection will benefit from this form of treatment. In the trial of Taylor et al [6], approximately 20% of liver recurrences occurred in the group in which portal vein perfusion had been performed. In the Australia and New Zealand Bowel Cancer Trial (a three-arm study), approximately 25% of hepatic relapses have occurred in this treatment arm (unpublished data). Because of the limited sensitivity of imaging techniques and intraoperative examination, it is likely that many patients who appear free of disseminated cancer in fact have liver metastases up to at least a few millimeters in diameter [4]. A likely reason for the aforementioned treatment failures is the inability of portal chemotherapy to destroy liver metastases beyond a certain stage of development. We have tested this hypothesis in an animal model. The aim of the study was to determine the relationship between the size of liver metastases at the time of treatment and the ability of regional chemotherapy to alter their progression. This relationship was determined for both portal vein and hepatic artery drug infusion. Experiments were conducted in a rat model of liver micrometastases generated by the introduction of cultured colonic carcinoma spheroids into the portal vein. MATERIAL AND M E T H O D S Inbred male Wistar WAG rats weighing between 250 and 280 g were obtained from the Animal Resources
THE AMERICAN JOURNAL OF SURGERY
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ARCHER AND GRAY
Figure 1. Cannulatlon of the gastroduodenal artery for long-term hepatic artery infusion. Arrow identifies the tip of a fine polyethylene catheter at the division of the common hepatic artery.
Center at Murdoch University, Western Australia. The animals were fed standard rat pellets and water ad libiturn. All experiments on animals were conducted in accordance with the guidelines of the National Health and Medical Research Council of Australia. A detailed description of the techniques of hepatic t u m o r induction has previously been reported [8]. In summary, a cell line (192 NRc) derived from a 1,2dimethylhydrazine-induced colonic carcinoma is grown on the surface of microspheres. The microspheres are positively charged polystyrene ion exchange resins 45 to 75 #m in diameter. Equal aliquots of sterile microspheres are incubated with RPMI-1640 media, penicillin 100 U / mL, streptomycin 100 u g / m L and 5% fetal bovine serum. Carcinoma cells form a firmly attached monolayer over the surface of each microsphere. After free tumor cells are removed by washing, the suspension of tumor spheroids is diluted to an appropriate concentration for injection. In this study, the portal vein of Wistar WAG rats was injected with 5 • 104 tumor spheroids to produce discrete hepatic micrometastases. Animals were then randomly assigned to receive either no treatment, portal vein 5-FU infusion, or hepatic artery 5-FU infusion. A previous study of the growth characteristics of this tumor model indicated that nodules became macroscopically visible on liver sections at 5 to 6 days after tumor inoculation. After 14 days, hepatic nodules had a mean diameter of 1.5 4- 0.1 mm (SE) [8]. In the current study, 11 animals were inoculated with tumor and did not receive chemotherapy. Subsequent growth of hepatic metastases in this group was determined in the manner described later. Portal vein infusion was carried out by cannulation of a branch of the superior mesenteric vein. Hepatic artery infusion was accomplished by introduction of a polyethylene catheter into the gastroduodenal artery (Dural Plastics and Engineering, Auburn, Australia) (Figure 1). Catheters were tunneled subcutaneously and exited through a small metallic disk sutured to the posterior cervical musculature. The catheter and disk were then 326
connected to an infusion swivel chamber via a protective wire sheath. This apparatus allowed rotation of the animal without occlusion of the catheter lumen. Drug infusions were begun on either day 0, 2, 4, or 6 after the time of tumor inoculation. Groups of eight animals received either hepatic artery or portal vein chemotherapy beginning each of these days. 5-FU was administered at a dose of 30 mg/kg/day in a volume of 10 m L / day 5% dextrose using a three-channel syringe pump (Harvard Apparatus Ltd., Kent, England). Infusions were maintained for a period of 7 days in each rat. Catheter position was confirmed either by aspiration of blood on day 7 of treatment or, failing this, by direct visualization at laparotomy at the time the rats were killed. Rats in which catheters had become dislodged were excluded from the study. At the termination of treatment, the disk apparatus was removed, the catheter was occluded, and the end was buried subcutaneously. All animals were examined at 30 days after tumor inoculation for the presence of liver metastases. A 50% solution of India ink was infused into the inferior vena cava to demarcate hepatic tumor nodules. Livers were removed and fixed in Fekete's solution (formalin, ethyl alcohol, acetic acid) and sectioned at 1-mm intervals. Tumor appeared as discrete white nodules on a dark parenchymal background. The cross-sectional area of liver metastases on consecutive liver slices was calculated, and the total tumor burden was expressed in mm 2. Six additional untreated animals were killed in pairs at 2, 4, and 6 days after tumor inoculation, and the diameter of macroscopic metastases was measured. Paraffin sections of the livers were stained with hematoxylin and eosin and examined with light microscopy. These animals were used to determine the mean diameter of hepatic metastases present at the time of beginning of drug infusions. The statistical significance of observed differences between control animals and the different treatment groups was determined using a Mann-Whitney test. RESULTS C o n t r o l animals: The total burden of liver metastases in 11 rats not treated with chemotherapy and killed 30 days after tumor inoculation was assessed in the aforementioned manner (Table I). The reproducibility of the model is reflected in the similarity of tumor growth between animals. The mean total cross-sectional area of metastases was 12,882 4- 653 mm 2 (SE). Regional infusion of 5-FU: Seven-day infusions were successfully completed in a total of 32 animals (16 portal vein; 16 hepatic artery). Figure 2 summarizes the results obtained in the treated animals. Both infusion routes demonstrated a significant reduction in liver metastases when 5-FU infusion was begun on the same day as tumor inoculation. The mean tumor burden in the portal vein group was 1,285 4- 980 mm 2 (SE). This represented a reduction of approximately 91% in liver metastases in comparison with the control group (p = 0.004). When hepatic artery infusion was begun immediately after tumor inoculation, the mean tumor growth was
THE AMERICAN JOURNAL OF SURGERY VOLUME159 MARCH 1990
REGIONAL CHEMOTHERAPY FOR HEPATIC MICROMETASTASES
TABLE I
16000-
Hepatic Tumor Growth in Control Animals
Animal No.
==
14000. .....
12OOO.
Tumor Burden at 30 Days (mm2)
10000' _>
1 2 3 4 5 6 7 8 9 10 11
13,122 15,047 14,616 11,391 10,969 14,145 9,866 13,279 14,903 9,238 15,125
CONTROLS
-=- PORTAL VEIN -e,., HEPATIC ARTERY
8000' ,~
6000 -
~Z
4000 2000
ch
8
o I
-
1
i
I
I
2
3
4
.
I
I
5
6
9
I
7
DAY INFUSION COMMENCED
Figure 2. Liver tumor burden at 1 month after inoculation follow-
1,906 4- 953 mm 2 (SE) (approximately 66% reduction; p 0.004). As the time of starting the portal vein infusion was delayed, the effectiveness of this treatment in reducing the subsequent growth of liver metastases diminished. When the infusion was begun 6 days after tumor implantation, there was no significant difference in tumor growth between the portal vein infusion group and the untreated group (p -- 0.36). In contrast, as hepatic artery infusion was delayed, its effectiveness was maintained. Final hepatic tumor growth after arterial infusions at 2, 4, and 6 days was significantly less than in the control group (p -- 0.004). There was no difference between the portal vein and hepatic artery treatment groups infused at either 2 or 4 days after tumor inoculation (p -- 0.56, respectively). However, at 6 days after implantation, hepatic artery 5FU was much more effective than portal vein chemotherapy in reducing liver metastases (p -- 0.02). H i s t o l o g y : Examination of rat livers at 2 and 4 days after tumor spheroid injection revealed no easily identifiable macroscopic liver metastases. In the two untreated animals examined at 6 days, a sharp contrast between tumor and normal liver was obtained using the India ink technique. Macroscopic lesions in sectioned livers at 6 days had a mean diameter of 0.33 4- 0.03 mm (SE) (n -65). These were subsequently examined histologically, and the range of lesions observed is demonstrated in Figures 3 and 4. The largest lesions (approximately 0.45 mm) were seen to be distending portal venules and early invasion had occurred outside the wall of the vessel (Figure 4).
ing either continuous hepatic artery or portal vein 5-FU (30 mg/ kg/day) infusion for 7 days. Each value is the mean 4- SD of data from four rats. Interrupted line represents the mean tumor growth in 11 control animals.
=
COMMENTS We have investigated how the timing of regional chemotherapy administered for liver micrometastases via either the portal vein or hepatic artery affects subsequent tumor growth. Experimental micrometastases were induced by the embolization of microspheres coated with a colonic carcinoma cell line into portal vein radicles. We conclude that the effectiveness of 5-FU administered into the portal vein diminishes as hepatic micrometastases expand within portal vein radicles. When malignant inva-
sion through the walls of these vessels has occurred, chemotherapy via the portal route does not influence disease progression. Macroscopic lesions at this stage of development have a mean diameter of 0.33 4- 0.03 mm (SE). The relationship between treatment response and tumor size was significantly different for the hepatic artery. Notably, when arterial infusions were started immediately after tumor inoculation, a significant reduction in liver metastases was achieved. Furthermore, this effect was maintained or slightly improved as treatment was delayed. The explanation for these findings lies in the development of tumor blood supply. We have previously demonstrated in this model that the portal vein plays only a passive role in the nutrition of small liver metastases [9]. Unlike the hepatic artery, which has been shown to perfuse metastases as small as 0.5 ram, the portal vein does not significantly vascularize liver metastases of any size. This implies that nutrition of liver micrometastases by the portal vein relies on diffusion of substances from sinusoids in the surrounding normal liver parenchyma. Similarly, cytotoxic drugs such as 5-FU infused into the portal vein would reach tumor tissue in this manner. As the diffusion of most chemotherapy drugs through tissue is minimal, tumor size is likely to be a major determinant of the effectiveness of intraportal treatment. Furthermore, branches of the portal vein at the tumor periphery become invaded and compressed during growth of metastases, diminishing drug delivery to tumor tissue even more [10]. In contrast, drugs delivered into the hepatic artery are targeted into tumors with a developing internal circulation. Consequently, an advantage for the arterial route over the portal vein should exist for vascularized metastases in the "adjuvant" setting. It is interesting to note that an approximately 66% reduction in liver metastases (compared with controls) was achieved in this experimental study when hepatic artery infusion of 5-FU was started immediately after tumor injection. This compared with an approximately 91% reduction in final tumor burden after portal vein
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Figure 3. Tumor adherent to the wall of portal venule 6 days after InjecUon of cancer spheroids (arrow) (hematoxylln-eosin stain, original rnagnlflcation X 180).
infusion (p = 0.04). Since no anatomic changes in the blood supply of these lesions have occurred at this stage, we can assume that significant drug diffusion occurred into tumor from arterially filled sinusoids in the normal liver parenchyma, in a similar manner to portal vein chemotherapy. Several clinical trials are in progress to assess the role of portal vein chemotherapy in the prevention of liver metastases from colonic carcinoma. They use 5-FU either alone or in combination with another drug [7,11]. T h e s e trials were established after a report by Taylor et al [6] in 1985 that indicated a significant improvement in survival for patients with stage B or C disease receiving 5FU via this route. The rationale for administering chemotherapy into the portal vein in the adjuvant setting is twofold. First, colonic carcinoma spreads by invasion of mesenteric veins and dissemination into portal venous blood. Surgery is thought to enhance this process of dissemination, as malignant cells have been isolated from portal blood in the perioperative period [12]. Portal vein chemotherapy might destroy these micrometastases before they develop into overt lesions. Second, qualitative studies in experimental models had suggested that a mixed portal-arterial circulation existed in small liver metastases, sometimes with predominance of the portal component [13]. It was hoped that portal vein chemotherapy could treat occult liver metastases up to a few millimeters in size. However, the results of our study indicate that once liver micrometastases invade the surrounding parenchyma, portal vein chemotherapy has little effect on their progression. This has important implications in the clinical setting. At best, liver imaging and intraoperative ex328
Figure 4. Tumor growing out from polystyrene mlcrospheres at 8 days. The nodule has distended the portal venule and early tumor invasion outside the vessel has occurred (arrow) (hematoxyllneos!n stain, original magnification X 180).
amination can only detect liver metastases as small as a few millimeters in diameter [4]. At worst, they may miss lesions of more than 2 cm in diameter, particularly in the posterior aspect of the right lobe of the liver. It follows that many patients with clinical stage B or C colonic carcinoma have established liver metastases at the time of surgery. These patients might be expected to respond poorly to intraportal chemotherapy. If the survival advantage from reduction of liver metastases is to be improved even further, a combination of treatment modalities is required, Our findings would suggest that the hepatic artery should be used as a complementary route of drug administration to portal vein chemotherapy. This hypothesis requires testing in a clinical trial. ACKNOWLEDGMENT We thank Mrs. A. Storrie and Mrs. J. Funk for their assistancewith the surgical proceduresin this study. We are also grateful to Dr. L. Matz for conducting the histology.
This experiment is well designed and could be helpful infurther protocol refinement. Both the timing and route of administration are interesting. REFERENCES 1. Willett CG, Tepper JE, Cohen AM, Orlow E, Welch CE. Failure patterns followingcurativeresectionof coloniccarcinoma. Ann Surg 1984; 6: 685-90. 2. Gilbert JM, Jeffrey I, Evans M, Kark AE. Sites of recurrent tumour after "curative" colorectal surgery: implications for adjuvant therapy9Br J Surg 1984; 71: 203-5. 3. BozzettiF, BignamiP, MorabitoA, Doci R, Gennari L. Patterns
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of failure following surgical resection of colorectal cancer liver metastases. Ann Surg 1987; 205: 264-70. 4. Finlay IG, McArdle CS. Occult hepatic metastases in colorectal carcinoma. Br J Surg 1986; 73: 732-5. 5. Buyse M, Zeleniuch-Jacquotte A, Chalmers TC. Adjuvant therapy of colorectal cancer: a meta-analysis of published clinical trials. Heidelberg: EORTC Symposium on Gastrointestinal Tract Cancer, 1986. 6. Taylor I, Machin D, Mullee M, Trotter G, Cooke T, West C. A randomized controlled trial of adjuvant portal vein cytotoxic perfusion in colorectal cancer. Br J Surg 1985; 72: 359-63. 7. Gray BN, De Zwart J, Fisher R, et al. The Australia and New Zealand trial of adjuvant chemotherapy in colon cancer. In: Jones S, Salmon SE, eds. Adjuvant therapy of cancer V. New York: Grune & Stratton, 1987: 537-46. 8. Archer SG, Gray BN. A new reproducible model of hepatic and peritoneal metastases from colonic carcinoma. Eur J Cancer Clin
Oncol 1988; 24: 1623-32. 9. Archer SG, Gray BN. Vascularization of small liver metastases. Br J Surg 1989; 76: 545-8. 10. Lin G, Lunderquist A, Hagerstrand I, Boijsen E. Postmortem examination of the blood supply and vascular pattern of small liver metastases in man. Surgery 1984; 96: 517-26. 11. Metzger U, Mermillod B, Aeberhard P, et al. Adjuvant portal liver infusion with 5-fluorouracil and mitomycin C followingcurative large bowel cancer surgery. In: Jones S, Salmon SE, eds. Adjuvant therapy of cancer IV. New York: Grune & Stratton, 1984: 471-8. 12. Wiggers T, Jeekel J, Arends JW, et al. No-touch isolation technique in colon cancer: a controlled prospective trial. Br J Surg 1988; 75: 409-15. 13. Ackerman NB. The blood supply of experimental liver metastases. IV. Changes in vascularity with increasing tumor growth. Surgery 1974; 4: 589-96.
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This study was conducted in a rat model of hepatic micrometastases generated by the intraportal injection of a colonic carcinoma cell line bound to polystyrene microspheres. Thirty-two animals received continuous infusions of 5-fluorouracil (5-FU) into either the portal vein or hepatic artery for a period of 7 days. Drug infusions were begun at O, 2, 4, and 6 days after the time of tumor inoculation in four groups of animals, respectively. Subsequent tumor growth in these animals at 1 month was compared with tumor growth in 11 control animals that did not receive chemotherapy. When 5-FU was administered via the portal vein On the same day as tumor inoculation, liver metastases were reduced by approximately 91% (p = 0 . 0 0 4 ) . Portal vein chemotherapy administered 6 days after tumor inoculation, when macroscopic nodules had a mean diameter of 0.33 + 0.03 mm ( S E ) , produced no tumor response (p = 0 . 3 6 ) . Histologic examination of these lesions revealed early invasion outside distended portal venules. In contrast, hepatic artery 5-FU infusions administered at 0, 2, 4, and 6 days after tumor implantation all reduced the subsequent development of hepatic metastases by approximately two thirds of that observed in the untreated group (p = 0 . 0 0 4 ) . We conclude that hepatic artery chemotherapy may have an important complementary role to play as an adjuvant treatment for gastrointestinal cancer.
From the University Department of Surgery, Royal Perth Hospital, Perth, Western Australia. Supported by the Royal Perth Hospital Research Foundation and the Royal Australasian College of Surgeons Research Foundation. Requests for reprints should be addressed to Stephen G. Archer, MBBS, University Department Of Surgery, Royal Perth Hospital, Perth, Western Australia, 6000. Manuscript submitted November 10, 1988, revised April 10, 1989, and accepted April 18, 1989.
n approximately 70% of patients with colorectal canItimecer, the disease appears limited to the bowel at the of surgical resection; in most patients, however, dissemination will occur. The liver is the most common site of recurrence, accounting for up to 30% of treatment failures [1-3]. Some hepatic recurrences may be attributed to the seeding of tumor cells into portal venous blood during and after surgical manipulation of the primary tumor. Others may represent the progression of established metastases that are clinically undetectable at the time of surgery [4]. Adjuvant systemic chemotherapy has been shown to be of little benefit in patients with operable bowel cancer [5]. However, two recent clinical trials have shown a significant survival advantage for patients receiving regional postoperative chemotherapy. The first of these trials, by Taylor et al [6], demonstrated a significant survival advantage for patients with stage B or C colonic carcinoma receiving 7 days of portal vein 5-fluorouracil (5-FU). Subsequently, the Australia and New Zealand Bowel Cancer Trial has confirmed the findings in stage C disease [7]. The survival advantage from portal vein administration of 5-FU primarily stems from a reduction in the incidence of liver metastases. However, not all patients with clinically normal livers at the time of resection will benefit from this form of treatment. In the trial of Taylor et al [6], approximately 20% of liver recurrences occurred in the group in which portal vein perfusion had been performed. In the Australia and New Zealand Bowel Cancer Trial (a three-arm study), approximately 25% of hepatic relapses have occurred in this treatment arm (unpublished data). Because of the limited sensitivity of imaging techniques and intraoperative examination, it is likely that many patients who appear free of disseminated cancer in fact have liver metastases up to at least a few millimeters in diameter [4]. A likely reason for the aforementioned treatment failures is the inability of portal chemotherapy to destroy liver metastases beyond a certain stage of development. We have tested this hypothesis in an animal model. The aim of the study was to determine the relationship between the size of liver metastases at the time of treatment and the ability of regional chemotherapy to alter their progression. This relationship was determined for both portal vein and hepatic artery drug infusion. Experiments were conducted in a rat model of liver micrometastases generated by the introduction of cultured colonic carcinoma spheroids into the portal vein. MATERIAL AND M E T H O D S Inbred male Wistar WAG rats weighing between 250 and 280 g were obtained from the Animal Resources
THE AMERICAN JOURNAL OF SURGERY
VOLUME 159 MARCH 1990
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ARCHER AND GRAY
Figure 1. Cannulatlon of the gastroduodenal artery for long-term hepatic artery infusion. Arrow identifies the tip of a fine polyethylene catheter at the division of the common hepatic artery.
Center at Murdoch University, Western Australia. The animals were fed standard rat pellets and water ad libiturn. All experiments on animals were conducted in accordance with the guidelines of the National Health and Medical Research Council of Australia. A detailed description of the techniques of hepatic t u m o r induction has previously been reported [8]. In summary, a cell line (192 NRc) derived from a 1,2dimethylhydrazine-induced colonic carcinoma is grown on the surface of microspheres. The microspheres are positively charged polystyrene ion exchange resins 45 to 75 #m in diameter. Equal aliquots of sterile microspheres are incubated with RPMI-1640 media, penicillin 100 U / mL, streptomycin 100 u g / m L and 5% fetal bovine serum. Carcinoma cells form a firmly attached monolayer over the surface of each microsphere. After free tumor cells are removed by washing, the suspension of tumor spheroids is diluted to an appropriate concentration for injection. In this study, the portal vein of Wistar WAG rats was injected with 5 • 104 tumor spheroids to produce discrete hepatic micrometastases. Animals were then randomly assigned to receive either no treatment, portal vein 5-FU infusion, or hepatic artery 5-FU infusion. A previous study of the growth characteristics of this tumor model indicated that nodules became macroscopically visible on liver sections at 5 to 6 days after tumor inoculation. After 14 days, hepatic nodules had a mean diameter of 1.5 4- 0.1 mm (SE) [8]. In the current study, 11 animals were inoculated with tumor and did not receive chemotherapy. Subsequent growth of hepatic metastases in this group was determined in the manner described later. Portal vein infusion was carried out by cannulation of a branch of the superior mesenteric vein. Hepatic artery infusion was accomplished by introduction of a polyethylene catheter into the gastroduodenal artery (Dural Plastics and Engineering, Auburn, Australia) (Figure 1). Catheters were tunneled subcutaneously and exited through a small metallic disk sutured to the posterior cervical musculature. The catheter and disk were then 326
connected to an infusion swivel chamber via a protective wire sheath. This apparatus allowed rotation of the animal without occlusion of the catheter lumen. Drug infusions were begun on either day 0, 2, 4, or 6 after the time of tumor inoculation. Groups of eight animals received either hepatic artery or portal vein chemotherapy beginning each of these days. 5-FU was administered at a dose of 30 mg/kg/day in a volume of 10 m L / day 5% dextrose using a three-channel syringe pump (Harvard Apparatus Ltd., Kent, England). Infusions were maintained for a period of 7 days in each rat. Catheter position was confirmed either by aspiration of blood on day 7 of treatment or, failing this, by direct visualization at laparotomy at the time the rats were killed. Rats in which catheters had become dislodged were excluded from the study. At the termination of treatment, the disk apparatus was removed, the catheter was occluded, and the end was buried subcutaneously. All animals were examined at 30 days after tumor inoculation for the presence of liver metastases. A 50% solution of India ink was infused into the inferior vena cava to demarcate hepatic tumor nodules. Livers were removed and fixed in Fekete's solution (formalin, ethyl alcohol, acetic acid) and sectioned at 1-mm intervals. Tumor appeared as discrete white nodules on a dark parenchymal background. The cross-sectional area of liver metastases on consecutive liver slices was calculated, and the total tumor burden was expressed in mm 2. Six additional untreated animals were killed in pairs at 2, 4, and 6 days after tumor inoculation, and the diameter of macroscopic metastases was measured. Paraffin sections of the livers were stained with hematoxylin and eosin and examined with light microscopy. These animals were used to determine the mean diameter of hepatic metastases present at the time of beginning of drug infusions. The statistical significance of observed differences between control animals and the different treatment groups was determined using a Mann-Whitney test. RESULTS C o n t r o l animals: The total burden of liver metastases in 11 rats not treated with chemotherapy and killed 30 days after tumor inoculation was assessed in the aforementioned manner (Table I). The reproducibility of the model is reflected in the similarity of tumor growth between animals. The mean total cross-sectional area of metastases was 12,882 4- 653 mm 2 (SE). Regional infusion of 5-FU: Seven-day infusions were successfully completed in a total of 32 animals (16 portal vein; 16 hepatic artery). Figure 2 summarizes the results obtained in the treated animals. Both infusion routes demonstrated a significant reduction in liver metastases when 5-FU infusion was begun on the same day as tumor inoculation. The mean tumor burden in the portal vein group was 1,285 4- 980 mm 2 (SE). This represented a reduction of approximately 91% in liver metastases in comparison with the control group (p = 0.004). When hepatic artery infusion was begun immediately after tumor inoculation, the mean tumor growth was
THE AMERICAN JOURNAL OF SURGERY VOLUME159 MARCH 1990
REGIONAL CHEMOTHERAPY FOR HEPATIC MICROMETASTASES
TABLE I
16000-
Hepatic Tumor Growth in Control Animals
Animal No.
==
14000. .....
12OOO.
Tumor Burden at 30 Days (mm2)
10000' _>
1 2 3 4 5 6 7 8 9 10 11
13,122 15,047 14,616 11,391 10,969 14,145 9,866 13,279 14,903 9,238 15,125
CONTROLS
-=- PORTAL VEIN -e,., HEPATIC ARTERY
8000' ,~
6000 -
~Z
4000 2000
ch
8
o I
-
1
i
I
I
2
3
4
.
I
I
5
6
9
I
7
DAY INFUSION COMMENCED
Figure 2. Liver tumor burden at 1 month after inoculation follow-
1,906 4- 953 mm 2 (SE) (approximately 66% reduction; p 0.004). As the time of starting the portal vein infusion was delayed, the effectiveness of this treatment in reducing the subsequent growth of liver metastases diminished. When the infusion was begun 6 days after tumor implantation, there was no significant difference in tumor growth between the portal vein infusion group and the untreated group (p -- 0.36). In contrast, as hepatic artery infusion was delayed, its effectiveness was maintained. Final hepatic tumor growth after arterial infusions at 2, 4, and 6 days was significantly less than in the control group (p -- 0.004). There was no difference between the portal vein and hepatic artery treatment groups infused at either 2 or 4 days after tumor inoculation (p -- 0.56, respectively). However, at 6 days after implantation, hepatic artery 5FU was much more effective than portal vein chemotherapy in reducing liver metastases (p -- 0.02). H i s t o l o g y : Examination of rat livers at 2 and 4 days after tumor spheroid injection revealed no easily identifiable macroscopic liver metastases. In the two untreated animals examined at 6 days, a sharp contrast between tumor and normal liver was obtained using the India ink technique. Macroscopic lesions in sectioned livers at 6 days had a mean diameter of 0.33 4- 0.03 mm (SE) (n -65). These were subsequently examined histologically, and the range of lesions observed is demonstrated in Figures 3 and 4. The largest lesions (approximately 0.45 mm) were seen to be distending portal venules and early invasion had occurred outside the wall of the vessel (Figure 4).
ing either continuous hepatic artery or portal vein 5-FU (30 mg/ kg/day) infusion for 7 days. Each value is the mean 4- SD of data from four rats. Interrupted line represents the mean tumor growth in 11 control animals.
=
COMMENTS We have investigated how the timing of regional chemotherapy administered for liver micrometastases via either the portal vein or hepatic artery affects subsequent tumor growth. Experimental micrometastases were induced by the embolization of microspheres coated with a colonic carcinoma cell line into portal vein radicles. We conclude that the effectiveness of 5-FU administered into the portal vein diminishes as hepatic micrometastases expand within portal vein radicles. When malignant inva-
sion through the walls of these vessels has occurred, chemotherapy via the portal route does not influence disease progression. Macroscopic lesions at this stage of development have a mean diameter of 0.33 4- 0.03 mm (SE). The relationship between treatment response and tumor size was significantly different for the hepatic artery. Notably, when arterial infusions were started immediately after tumor inoculation, a significant reduction in liver metastases was achieved. Furthermore, this effect was maintained or slightly improved as treatment was delayed. The explanation for these findings lies in the development of tumor blood supply. We have previously demonstrated in this model that the portal vein plays only a passive role in the nutrition of small liver metastases [9]. Unlike the hepatic artery, which has been shown to perfuse metastases as small as 0.5 ram, the portal vein does not significantly vascularize liver metastases of any size. This implies that nutrition of liver micrometastases by the portal vein relies on diffusion of substances from sinusoids in the surrounding normal liver parenchyma. Similarly, cytotoxic drugs such as 5-FU infused into the portal vein would reach tumor tissue in this manner. As the diffusion of most chemotherapy drugs through tissue is minimal, tumor size is likely to be a major determinant of the effectiveness of intraportal treatment. Furthermore, branches of the portal vein at the tumor periphery become invaded and compressed during growth of metastases, diminishing drug delivery to tumor tissue even more [10]. In contrast, drugs delivered into the hepatic artery are targeted into tumors with a developing internal circulation. Consequently, an advantage for the arterial route over the portal vein should exist for vascularized metastases in the "adjuvant" setting. It is interesting to note that an approximately 66% reduction in liver metastases (compared with controls) was achieved in this experimental study when hepatic artery infusion of 5-FU was started immediately after tumor injection. This compared with an approximately 91% reduction in final tumor burden after portal vein
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Figure 3. Tumor adherent to the wall of portal venule 6 days after InjecUon of cancer spheroids (arrow) (hematoxylln-eosin stain, original rnagnlflcation X 180).
infusion (p = 0.04). Since no anatomic changes in the blood supply of these lesions have occurred at this stage, we can assume that significant drug diffusion occurred into tumor from arterially filled sinusoids in the normal liver parenchyma, in a similar manner to portal vein chemotherapy. Several clinical trials are in progress to assess the role of portal vein chemotherapy in the prevention of liver metastases from colonic carcinoma. They use 5-FU either alone or in combination with another drug [7,11]. T h e s e trials were established after a report by Taylor et al [6] in 1985 that indicated a significant improvement in survival for patients with stage B or C disease receiving 5FU via this route. The rationale for administering chemotherapy into the portal vein in the adjuvant setting is twofold. First, colonic carcinoma spreads by invasion of mesenteric veins and dissemination into portal venous blood. Surgery is thought to enhance this process of dissemination, as malignant cells have been isolated from portal blood in the perioperative period [12]. Portal vein chemotherapy might destroy these micrometastases before they develop into overt lesions. Second, qualitative studies in experimental models had suggested that a mixed portal-arterial circulation existed in small liver metastases, sometimes with predominance of the portal component [13]. It was hoped that portal vein chemotherapy could treat occult liver metastases up to a few millimeters in size. However, the results of our study indicate that once liver micrometastases invade the surrounding parenchyma, portal vein chemotherapy has little effect on their progression. This has important implications in the clinical setting. At best, liver imaging and intraoperative ex328
Figure 4. Tumor growing out from polystyrene mlcrospheres at 8 days. The nodule has distended the portal venule and early tumor invasion outside the vessel has occurred (arrow) (hematoxyllneos!n stain, original magnification X 180).
amination can only detect liver metastases as small as a few millimeters in diameter [4]. At worst, they may miss lesions of more than 2 cm in diameter, particularly in the posterior aspect of the right lobe of the liver. It follows that many patients with clinical stage B or C colonic carcinoma have established liver metastases at the time of surgery. These patients might be expected to respond poorly to intraportal chemotherapy. If the survival advantage from reduction of liver metastases is to be improved even further, a combination of treatment modalities is required, Our findings would suggest that the hepatic artery should be used as a complementary route of drug administration to portal vein chemotherapy. This hypothesis requires testing in a clinical trial. ACKNOWLEDGMENT We thank Mrs. A. Storrie and Mrs. J. Funk for their assistancewith the surgical proceduresin this study. We are also grateful to Dr. L. Matz for conducting the histology.
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