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Comparative toxicity of chemotherapy following partial hepatectomy
Comparative Toxicity of Chemotherapy Following Partial Hepatectomy By Jay L. Grosfeld, Thomas R. Weber, and Robert L. Baehner Indianapolis, Indiana 9 Recent reports document an improved survival in children treated with chemotherapy following hepatic resection for malignant liver tumors. This report compares the effect of commonly employed chemotherapeutic agents on the regenerating liver. INDEX WORDS: Hepatic tumors; liver regeneration; chemotherapy toxicity.
mental strategy identifies selective agents that could be safely administered in the immediate to early posthepatectomy period when the growth fraction of susceptible residual tumor ceils is high and more vulnerable to cell cycle specific chemotherapy. METHODS
OMBINED cancer therapy (including courses of multiple agent chemotherapy) has played a significant role in improving the survival rate of infants and children with certain embryonal neoplasms. In children with primary liver malignancies hepatic lobectorny alone has resulted in survival in less than half the cases, 1'2 even when the tumor was completely resected. More recently, improved survival has been seen in additional patients after administering chemotherapeutic agents shown to be cytotoxic against hepatoblastoma and hepatocellular carcinoma. It has long been recognized that radiation may have a deleterious effect on both the intact and regenerating liver. 3-5 Certain chemotherapeutic agents are radiomimetic and can impair liver regeneration following hepatic lobectomy, resulting in considerable delays in instituting treatment. This study evaluates the impact of three tumoricidal agents on the synthetic and reciprocal catabolic pathways of thymidine metabolism as well as mitotic indices and organ weight of regenerating liver tissue in rats following 70% hepatectomy. This experi-
C
From the Section of Pediatric Surgery, Department of Surgery and the Section of Hematology-Oncology, Department of Pediatrics, lndiana University School of Medicine and the James Whitcomb Riley Hospital for Children, Indianapolis, Ind. Presented before the Twelfth Annual Meeting of the American Pediatric Surgical Association together with the British Association of Paediatric Surgeons, Tarpon Springs, Florida, April 29-May 2, 1981. Address reprint requests to Jay L. Grosfeld, M~D., Surgeon-in-Chief, J. IV. Riley Hospital for Children (K-21), 1100 W. Michigan Street, Indianapolis, Ind. 46223. 9 1981 by Grune & Stratton, Inc. 00 2 2-3 468/81/1606~0 355 01.00/0
950
Animals were kept in individual stainless steel cages and were fed standard rat chow and water ad libitum. A standardized 70% hepatectomy was performed in 200 SpragueDawley rats weighing 175 g using sterile technique and ether anesthesia. Animals were divided into four experimental groups. Group I (n = 50) acted as untreated controls, group II (n = 50)received Actin0mycin-D ( A M D ) 150 ~tg/kg i . v . , group III (n = 50) Vincristine (VCR) 0.2 m g / k g i.v. and group IV (n = 50) Adriamycin ( A D R ) 30 m g / s q m i.v. Opposing metabolic pathways (thymidine incorporation to D N A and thymidine degradation to CO2) were measured in liver slices using thymidine 2-14Cat 0, 18, 24, 30, and 48 hr following hepatectomy. The number of mitosis per high power field (HPF) at 24 hr were evaluated in each group. Liver weight was determined at 1 wk following partial hepatectomy. Rats were stunned, decapitated, and exsanguinated. Metabolic studies were carried out according to the methods of Ferdinandus et al. 6 Livers were rapidly removed and placed in beakers which stood on crushed ice. The tissues were sectioned into 1.0-2.0 cm cubes and slices were cut to a uniform thickness of 1.0-2.0 m m by a Stadie-Riggs slicer. Tissue slices (50 rag) were placed into separate 25.0 ml Erlenmeyer flasks each containing 5.0 ml of Krebs-RingersTris buffer (pH 7.4), 118 m M NaCI, 25 m M Tris-HCl, 4.7 m M KCI, 2.5 m M CaCI2, 1.2 m M MgSO4, 1.2 m M KHzPO4 and 0.5 t~Ci of thymidine-2-14C (specific activity 58.9 u C i / m m o l e - - N e w England Nuclear). Each flask was capped with a rubber stopper from which was suspended a plastic well with a glass filter wick. Flasks were incubated at 37~ with shaking. The reaction was terminated after 0 and 30 min by injecting 1.0 ml of 50% trichloroacetic acid (TCA) into the bottom of the flask. CO2 was collected by injecting 0.2 ml of hyamine hydroxide into the well saturating the wick. The flasks were shaken an additional 30 min at 37~ The rubber stoppers were removed and the plastic wells and wicks were placed in scintillation vials. Ten milliliters of scintillator fluid was immediately added to each vial and measured in a scintillator counter. The remaining contents of the flasks were homogenized, transferred to 10 ml volumetric flasks and brought to volume with distilled water. The volumetric flasks were shaken vigorously for 30 see and 0.5 ml aliquots were pipetted into a separate mil!ipore filter apparatus containing a glass filter
Journal of Pediatric Surgery, VoI. 16, No. 6 (December),1981
CHEMOTHERAPY TOXICITY FQLLOW~NG HEPATECTOMY
951
disc and 5 ml of ice cold 10% TCA. Suction was applied and the filter disc was washed with 100% ice cold ethanol. The filters were ovendried at 38~ for 1 hr and placed in 10 ml of scintillation fluid and counted. Liver sections were stained with hemotoxylin and eosin and the number of mitoses per high per field at 24 hr were counted. Liver weight (10 animals in each group) was evaluated at 1 wk.
Table 1. Liver W t and No. of M i t o s e s / H P F in Four Experimental Groups Group I (Control) IIAMB III VCR IVADR
Liver (wt)
Mistosis/HPF No.
10.42 -+ 1.26 7.21 -+ 2.26
4-5 0-1
9.33 -+ 1.37 5.12 +_ 1.41
2-3 0
RESULTS
In controls (group I), the peak D N A levels (8.2 _+ 0.6 • 103 C P M ) and the lowest CO2 levels were seen at 24 hr. Vincristine (group I I I ) had little effect on the regenerating liver with D N A synthesis (7.92 _+ 1.2 x 203 C P M ) and CO2 degradation activity similar to untreated controls. D N A levels in group IV ( A D R ) was 5.1 + 0.8 • 103 C P M and was lowest in group II animals (AMD) at 3.78 + 0.86 • 103 C P M (p < 0.01) (Fig. 1). The highest levels of CO2 degradation was seen in groups II and IV. Liver weight at 1 wk was 10.42 _+ 1.26 g in group I controls, 9.33 + 1.37 g in group II] (VCR), 7.21 _+ 2.26 g in group lI ( A M D ) and only 5.12 _+ 1.41 g in group IV ( A D R ) (p < 0.01). The number of m i t o s e s / H P F at 24
THYMIDINE-2-C14 15-
9 DNA
O~-OADR ~-
1098xl0 a
I 12
'
I
24
I
I
48
72
HOURS after partial hepatectomy Fig. 1 This illustration demonstrates that incorporat i o n of thymidine-2 14C into DNA is similar in Vincristine treated and control rats. Actinomycin-D and Adriamycin significantly (p < 0.O1 ) i n t e r f e r e w i t h D N A synthesis.
hr was 4 - 5 / H P F in controls, 2 - 3 / H P F in V C R (group III) rats, 0 - 1 / H P F in A M D (group II) and zero in A D R (group IV) (Table 1). These data indicate both A M D and A D R severely inhibit D N A synthesis. Adriamycin treated rats had the lowest liver weights and number of mitoses per high power field. Vincristine was a relatively safe agent being similar to controls in regard to D N A synthesis, liver weight, and progress of regeneration. DISCUSSION
The best survival in primary liver tumors in children is seen with hepatic lobectomy done for cure. ''2 In many cases, however, there may be extension of gross or microscopic residual tumor beyond the margin of anatomic resection or unknown micrometastases may already have occurred, particularly to the lung. Under these latter circumstances chemotherapy may play a role in improving the overall survival rate. There is also some evidence to suggest that multiple agent chemotherapy may shrink a liver tumor and change it from an inoperable to an operable neoplasm.' Recent data from the Children's Cancer Study Group demonstrate an improved survival in cases of stage I and II hepatic neoplasms with adjunctive chemotherapy if resection can be achieved. Currently the agents used most frequently are Adriamycin, Vincristine, 5-fluorouracil, cyclophosphamide, cis-platihum and bleomycin. Chemotherapy and radiation may have injurious effects on both the intact and regenerating liver. A number of reports demonstrate that radiation and chemotherapy may acutely cause severe hepatic vascular congestion with resultant edema, subendothelial hemorrhage, and fibrous necrosis of sublobular veins. 3-5 Hepatomegaly is often observed and is associated with serum e n z y m e elevations ( a l k a l i n e p h o s p h a t a s e , SGOT). Radioisotopic liver scans show an irreg-
952
ular distribution of the isotope within the irradiated portions of the liver and increased pickup of isotope in the spleen. 7 Severe liver dysfunction characterized by jaundice, ascites, hypoalbuminemia and prolonged prothrombin time has been seen in some cases with an occasional mortality noted. These latter findings are unusual unless the liver is treated with >3500 rad, but has also been observed with as little as 2000 rad when chemotherapy is also employed.4'7 The chronic phase of radiation or drug induced hepatitis is characterized by fibrosis of portal and perivascular portions of the previously irradiated liver. The previously irradiated area of liver becomes small and shrunken while the unirradiated portion of the liver is usually enlarged because of compensatory hypertrophy. 3'7 Filler et al. reported a 70% mortality in rats following partial hepatectomy when treated with Actinomycin-D and irradiation. 3 This was: compared to a 5% mortality for partial hepatectomy alone and a 12% mortality in rats with intact livers treated with similar doses of chemotherapy and irradiation. A number of other authors have shown that liver regeneration is inhibited by both radiation and chemotherapyP '8 These treatment modalities suppress those enzymes responsible for DNA and R N A synthesis in residual liver tissue. These findings have led to significant clinical delays (up to 1 mo) in initiating chemotherapy programs in patients with hepatic neoplasms following partial hepatectomy. Liver regeneration is characterized by a wave of proliferative activity that involves the parenchymal cells (hepatocytes) almost exclusively. DNA synthesis usually begins at 15-18 hr and reaches a peak level at 24-26 hr. 9 Bile duct and supportive tissue cells enter DNA synthesis and mitosis much later; a maximal rate of DNA formation is not achieved until 36-42 hr posthepatectomy. Livers of younger animals (newborn or weanling rats) are restored more rapidly than in the adult animal. 9 In addition, a second wave of DNA activity occurs in weanling rats at 33-35 hr and represents proliferation of both parenchymal and nonparenchymal cells. 9 This probably indicates a relatively greater stimulation in weanling animals in which hepatic regeneration is superimposed upon an already rapidly
GROSFELD, WEBER, AND BAEHNER
growing liver. Under these conditions, presumably a larger portion of cells would have to divide repetitively during the first few days to compensate for the regenerative demand as well as to continue normal growth. These experimental findings are supported by the clinical observations in young children who have undergone partial hepatectomy. Sequential liver scans demonstrate rather rapid liver regeneration; postoperative metabolic parenteral support (adjunctive glucose, albumin, vitamin-K) is required for only a few days. Partial hepatectomy results in marked changes in the behavior of tbymidine metabolism. The activity of the synthetic pathway (incorporation to DNA) increases while the activity of the catabolic pathway (degradation to CO2) decreases. 6 Therefore, study of these opposing pathways are very useful to evaluate hepatic differentiation, regeneration and the effect of antimetabolites on the liver cell. In addition, these metabolic parameters are useful in studying the cellular metabolism of tumors. 6 Murthy et al. demonstrated that the nucleic acid and protein content of liver tumors in children are related to nucleocytoplasmic ratios and histologic maturity of the tumor. ~~ They noted that the DNA content of hepatoblastomas exceeded those of control livers. However, DNA content of hepatocarcinoma was similar to that of control liver content. Ferdinandes et al. 6 observed that incorporating thymidine 2-~4C into DNA increases and concurrently the degradation to CO2 decreases parallel with hepatoma growth in rats. The ratio of synthetic:catabolic pathways correlated directly with hepatoma growth. This imbalance was actually more pronounced than in the regenerating liver. They concluded that this marked imbalance in thymidine metabolism was linked with tumor growth but may also be related to a neoplastic alteration of gene expression. Labow et al. H theorized that the observed rise in enzyme synthesis following partial hepatectomy resulted from either a depression or induction of closely linked genes. These "forces" were considered capable of restoring the balance of the steady liver state after the wave of hepatic regeneration reached completion. This was signified by reestablishing the number of parenchymal cells and
CHEMOTHERAPY TOXICITY FOLLOWING HEPATECTOMY
953
restoring preoperative liver weight and liver:body ratio. This report confirms previous observations concerning the deleterious effects of Actinomycin-D on the regenerating liver. 3'5'6ActinomycinD combines with deoxyguanine residues to inhibit DNA-directed RNA synthesis. As expected, in rapidly proliferating liver cells, RNA mediated protein synthesis as well as nuclear division is impaired. Similarly, the anthracycline, Adriamycin, also impairs the synthetic capacities of cells in active cycle by blocking DNA biosynthesis by forming complexes directly with DNA. These data demonstrate that adriamycin also significantly inhibits DNA synthesis and interferes with liver regeneration. The lowest liver weights and least numbers of mitosis/HPF were observed using this drug. Previous data from our laboratory indicated that vincristine may have a protective effect on the intact liver exposed to the damaging effect of Actinomycin-D and irradiation and prevent the onset of radiation hepatitis. 5 In contrast to
Actinomycin-D and Adriamycin, Vincristine exerts its effect on cell division by binding to tubulin, the subunit structures required for microtubules to polymerize into spindle threads. This drug merely blocks mitosis but has no effect on DNA, RNA, or protein synthesis. Additional studies evaluating Vincristine in rats with 70% partial hepatectomy suggest that this drug does little to interfere with hepatic regeneration. 8 The present report confirms this latter finding. Following partial hepatectomy, Vincristine treated animals were similar to controls in regard to thymidine incorporation into DNA, liver weight and overall progress of regeneration. Our observations in this study indicate that Adriamycin and Actinomycin-D should be withheld until liver regeneration is near completion. The data also suggest that Vincristine may prove useful in the early posthepatectomy state when there is suspected microscopic residual disease or incomplete resection since it has a less injurious effect on regenerating residual liver tissue.
REFERENCES
1. Exelby PR Filler RM, and Grosfeld JL: Liver tumors in children in the particular reference to hepatoblastoma and hepatocellular carcinoma. J Pediatr Surg 10:329-337, 1975 2. Clatworthy HW, Schiller M, Grosfeld JL: Primary liver tumors in infancy and childhood: 41 cases variously treated. Arch Surg 109:143-147, 1974 3. Filler RM, Tefft M, Vawter GF, et al: Hepatic lobectomy in childhood: Effect of x-ray and chemotherapy. J Pediatr Surg 4:31-41, 1969 4. Ingold JA, Reed GB, Kaplan HS, et al: Radiation hepatitis. Am J Roentgenol 93:200-208, 1965 5. Grosfeld JL, Cooney DR, Shidnia H, et al: The effect of combined cancer therapy on the intact liver. Surg Forum 26:426-428, 1975 6. Ferdinandus JA, Morris HP, Weber G: Behavior of opposing pathways of thymidine utilization in differentiat-
ing, regenerating and neoplastic liver. Cancer Res 31:550556, 1971 7. Samuels LD, Grosfeld JL, Kartha M: Radiation hepatitis in children. J Pediatr 78:68 73, 1971 8. Grosfeld JL, Ferdinandus L, Cakmak O, et al: Safe use of Vincristine following partial hepatectomy. Surg Forum 27:89-91, 1976 9. Bucher NLR, Swaffield MN, Ditroia JF: The influence of age upon the incorporation of thymidine 2-~4C into DNA of regenerating rat liver. Cancer Res 24:509-512, 1964 10. Murthy ASK, Vawter GF, Kipito L, et al~ Biochemical studies on liver tumors of children. Arch Pathol 96:4852, 1973 11. Labow R, Maley GF, Maley F: The effect of methotrexate on enzymes induced following partial hepatectomy. Cancer Res 29:366-372, 1969
954
GROSFELD, WEBER, AND BAEHNER
Discussion Daniel M. Hays (Los Angeles): Five years ago we were not sure if adjuvant chemotherapy played any role in managing patients with complete hepatic tumor resection. Today I think we can say that adjuvant therapy does clearly play a role. The current CCSG study demonstrates that patients with either hepatoblastoma and hepatocarcinoma have a 2-yr survival rate of 90% when the tumor is completely excised if they receive a four-drug chemotherapy regimen for 1 yr. Dr. Grosfeld's study is of particular importance because all of the chemotherapeutic systems should work more effectively early in the postexcision interval. The aim is to try and find the earliest time at which the chemotherapy can be started. I like Dr. Grosfeld's concept that liver regeneration need not be complete before starting chemotherapy. I have calculated the vincristine dosage as three times the amount that one would give in human medicine; the dactinomycin as about two times the human dose; and the adriamycin as about equal to the human dose, so he has given sufficient vincristine. I do not think one should infer that adriamycin is less hepatotoxic than actinomycin D; it is probably more
others (notably in this study, Vincristine) do not cause these problems. The clinical implications of this study are quite clear. We must know the action of each chemical agent on hepatic regeneration before its use in close proximity to surgery. The same applies to radiation therapy. We have previously reported studies with radiation therapy and Actinomycin D and their effect on hepatic regeneration. An additional aspect of this problem worthy of consideration is that these chemotherapeutic agents are ordinarily detoxified in the liver; therefore, when hepatic resection is performed, detoxification is impaired. Safe doses when the liver is intact may prove to be toxic after hepatecomy. Thus, the drug not only limits regeneration, but resection limits drug detoxification. One question I would ask Dr. Grosfeld is whether he has any information about the effect of these agents before hepatic resection? We are all familiar with recent reports about preoperative chemotherapy in children with hepatic tumors for "inoperable" tumors. I wonder if this approach might also limit liver regeneration.
SO.
Jay Grosfeld (Closure): I think Dr. Filler's question certainly is of interest. Actually, there is some correlary between hepatoma growth and the D N A synthetic pathway. The D N A synthetic rate of hepatoblastoma is similar to the regenerating liver and suggests that this tumor should be very susceptible to preoperative chemotherapy. This observation lends credence to Dr. Vos' comments regarding shrinking a large tumor in selected cases. Evaluating thymidine metabolism in the liver tumors may yield a prognostic factor. If the tumor has the ability to incorporate D N A it should be more responsive to chemotherapy. We plan to also study the effect of other drugs such as Bleomycin, Cytoxan and 5FU, (currently being employed in patients with liver tumors) on thymidine metabolism. Dr. Hays' comments were very kind. There were no statistical differences observed between adriamycin and actinomycin so what he said is absolutely correct.
Anton Vos (Netherlands): At the Children's Hospital in Amsterdam we recently have adopted a regimen using preoperative chemotherapy. We have treated three children with angiographic evidence of both liver lobes involved with hepatoblastoma. After several courses of chemotherapy (in one case over a total period of 10 mo) repeat angiography showed that the tumor was restricted to one lobe, which was subsequently resected. All three children are 2 yr postoperation, are free of disease and do not receive any chemotherapy. What Dr. Hays was talking about is the regeneration after the operation. Our experience is that on the liver scan made 6 mo after operation, the size of the liver had returned to normal. R.M. Filler (Toronto): This study gives both biochemical and morphological evidence that certain chemotherapeutic agents inhibit liver regeneration after hepatic resection, whereas
mental strategy identifies selective agents that could be safely administered in the immediate to early posthepatectomy period when the growth fraction of susceptible residual tumor ceils is high and more vulnerable to cell cycle specific chemotherapy. METHODS
OMBINED cancer therapy (including courses of multiple agent chemotherapy) has played a significant role in improving the survival rate of infants and children with certain embryonal neoplasms. In children with primary liver malignancies hepatic lobectorny alone has resulted in survival in less than half the cases, 1'2 even when the tumor was completely resected. More recently, improved survival has been seen in additional patients after administering chemotherapeutic agents shown to be cytotoxic against hepatoblastoma and hepatocellular carcinoma. It has long been recognized that radiation may have a deleterious effect on both the intact and regenerating liver. 3-5 Certain chemotherapeutic agents are radiomimetic and can impair liver regeneration following hepatic lobectomy, resulting in considerable delays in instituting treatment. This study evaluates the impact of three tumoricidal agents on the synthetic and reciprocal catabolic pathways of thymidine metabolism as well as mitotic indices and organ weight of regenerating liver tissue in rats following 70% hepatectomy. This experi-
C
From the Section of Pediatric Surgery, Department of Surgery and the Section of Hematology-Oncology, Department of Pediatrics, lndiana University School of Medicine and the James Whitcomb Riley Hospital for Children, Indianapolis, Ind. Presented before the Twelfth Annual Meeting of the American Pediatric Surgical Association together with the British Association of Paediatric Surgeons, Tarpon Springs, Florida, April 29-May 2, 1981. Address reprint requests to Jay L. Grosfeld, M~D., Surgeon-in-Chief, J. IV. Riley Hospital for Children (K-21), 1100 W. Michigan Street, Indianapolis, Ind. 46223. 9 1981 by Grune & Stratton, Inc. 00 2 2-3 468/81/1606~0 355 01.00/0
950
Animals were kept in individual stainless steel cages and were fed standard rat chow and water ad libitum. A standardized 70% hepatectomy was performed in 200 SpragueDawley rats weighing 175 g using sterile technique and ether anesthesia. Animals were divided into four experimental groups. Group I (n = 50) acted as untreated controls, group II (n = 50)received Actin0mycin-D ( A M D ) 150 ~tg/kg i . v . , group III (n = 50) Vincristine (VCR) 0.2 m g / k g i.v. and group IV (n = 50) Adriamycin ( A D R ) 30 m g / s q m i.v. Opposing metabolic pathways (thymidine incorporation to D N A and thymidine degradation to CO2) were measured in liver slices using thymidine 2-14Cat 0, 18, 24, 30, and 48 hr following hepatectomy. The number of mitosis per high power field (HPF) at 24 hr were evaluated in each group. Liver weight was determined at 1 wk following partial hepatectomy. Rats were stunned, decapitated, and exsanguinated. Metabolic studies were carried out according to the methods of Ferdinandus et al. 6 Livers were rapidly removed and placed in beakers which stood on crushed ice. The tissues were sectioned into 1.0-2.0 cm cubes and slices were cut to a uniform thickness of 1.0-2.0 m m by a Stadie-Riggs slicer. Tissue slices (50 rag) were placed into separate 25.0 ml Erlenmeyer flasks each containing 5.0 ml of Krebs-RingersTris buffer (pH 7.4), 118 m M NaCI, 25 m M Tris-HCl, 4.7 m M KCI, 2.5 m M CaCI2, 1.2 m M MgSO4, 1.2 m M KHzPO4 and 0.5 t~Ci of thymidine-2-14C (specific activity 58.9 u C i / m m o l e - - N e w England Nuclear). Each flask was capped with a rubber stopper from which was suspended a plastic well with a glass filter wick. Flasks were incubated at 37~ with shaking. The reaction was terminated after 0 and 30 min by injecting 1.0 ml of 50% trichloroacetic acid (TCA) into the bottom of the flask. CO2 was collected by injecting 0.2 ml of hyamine hydroxide into the well saturating the wick. The flasks were shaken an additional 30 min at 37~ The rubber stoppers were removed and the plastic wells and wicks were placed in scintillation vials. Ten milliliters of scintillator fluid was immediately added to each vial and measured in a scintillator counter. The remaining contents of the flasks were homogenized, transferred to 10 ml volumetric flasks and brought to volume with distilled water. The volumetric flasks were shaken vigorously for 30 see and 0.5 ml aliquots were pipetted into a separate mil!ipore filter apparatus containing a glass filter
Journal of Pediatric Surgery, VoI. 16, No. 6 (December),1981
CHEMOTHERAPY TOXICITY FQLLOW~NG HEPATECTOMY
951
disc and 5 ml of ice cold 10% TCA. Suction was applied and the filter disc was washed with 100% ice cold ethanol. The filters were ovendried at 38~ for 1 hr and placed in 10 ml of scintillation fluid and counted. Liver sections were stained with hemotoxylin and eosin and the number of mitoses per high per field at 24 hr were counted. Liver weight (10 animals in each group) was evaluated at 1 wk.
Table 1. Liver W t and No. of M i t o s e s / H P F in Four Experimental Groups Group I (Control) IIAMB III VCR IVADR
Liver (wt)
Mistosis/HPF No.
10.42 -+ 1.26 7.21 -+ 2.26
4-5 0-1
9.33 -+ 1.37 5.12 +_ 1.41
2-3 0
RESULTS
In controls (group I), the peak D N A levels (8.2 _+ 0.6 • 103 C P M ) and the lowest CO2 levels were seen at 24 hr. Vincristine (group I I I ) had little effect on the regenerating liver with D N A synthesis (7.92 _+ 1.2 x 203 C P M ) and CO2 degradation activity similar to untreated controls. D N A levels in group IV ( A D R ) was 5.1 + 0.8 • 103 C P M and was lowest in group II animals (AMD) at 3.78 + 0.86 • 103 C P M (p < 0.01) (Fig. 1). The highest levels of CO2 degradation was seen in groups II and IV. Liver weight at 1 wk was 10.42 _+ 1.26 g in group I controls, 9.33 + 1.37 g in group II] (VCR), 7.21 _+ 2.26 g in group lI ( A M D ) and only 5.12 _+ 1.41 g in group IV ( A D R ) (p < 0.01). The number of m i t o s e s / H P F at 24
THYMIDINE-2-C14 15-
9 DNA
O~-OADR ~-
1098xl0 a
I 12
'
I
24
I
I
48
72
HOURS after partial hepatectomy Fig. 1 This illustration demonstrates that incorporat i o n of thymidine-2 14C into DNA is similar in Vincristine treated and control rats. Actinomycin-D and Adriamycin significantly (p < 0.O1 ) i n t e r f e r e w i t h D N A synthesis.
hr was 4 - 5 / H P F in controls, 2 - 3 / H P F in V C R (group III) rats, 0 - 1 / H P F in A M D (group II) and zero in A D R (group IV) (Table 1). These data indicate both A M D and A D R severely inhibit D N A synthesis. Adriamycin treated rats had the lowest liver weights and number of mitoses per high power field. Vincristine was a relatively safe agent being similar to controls in regard to D N A synthesis, liver weight, and progress of regeneration. DISCUSSION
The best survival in primary liver tumors in children is seen with hepatic lobectomy done for cure. ''2 In many cases, however, there may be extension of gross or microscopic residual tumor beyond the margin of anatomic resection or unknown micrometastases may already have occurred, particularly to the lung. Under these latter circumstances chemotherapy may play a role in improving the overall survival rate. There is also some evidence to suggest that multiple agent chemotherapy may shrink a liver tumor and change it from an inoperable to an operable neoplasm.' Recent data from the Children's Cancer Study Group demonstrate an improved survival in cases of stage I and II hepatic neoplasms with adjunctive chemotherapy if resection can be achieved. Currently the agents used most frequently are Adriamycin, Vincristine, 5-fluorouracil, cyclophosphamide, cis-platihum and bleomycin. Chemotherapy and radiation may have injurious effects on both the intact and regenerating liver. A number of reports demonstrate that radiation and chemotherapy may acutely cause severe hepatic vascular congestion with resultant edema, subendothelial hemorrhage, and fibrous necrosis of sublobular veins. 3-5 Hepatomegaly is often observed and is associated with serum e n z y m e elevations ( a l k a l i n e p h o s p h a t a s e , SGOT). Radioisotopic liver scans show an irreg-
952
ular distribution of the isotope within the irradiated portions of the liver and increased pickup of isotope in the spleen. 7 Severe liver dysfunction characterized by jaundice, ascites, hypoalbuminemia and prolonged prothrombin time has been seen in some cases with an occasional mortality noted. These latter findings are unusual unless the liver is treated with >3500 rad, but has also been observed with as little as 2000 rad when chemotherapy is also employed.4'7 The chronic phase of radiation or drug induced hepatitis is characterized by fibrosis of portal and perivascular portions of the previously irradiated liver. The previously irradiated area of liver becomes small and shrunken while the unirradiated portion of the liver is usually enlarged because of compensatory hypertrophy. 3'7 Filler et al. reported a 70% mortality in rats following partial hepatectomy when treated with Actinomycin-D and irradiation. 3 This was: compared to a 5% mortality for partial hepatectomy alone and a 12% mortality in rats with intact livers treated with similar doses of chemotherapy and irradiation. A number of other authors have shown that liver regeneration is inhibited by both radiation and chemotherapyP '8 These treatment modalities suppress those enzymes responsible for DNA and R N A synthesis in residual liver tissue. These findings have led to significant clinical delays (up to 1 mo) in initiating chemotherapy programs in patients with hepatic neoplasms following partial hepatectomy. Liver regeneration is characterized by a wave of proliferative activity that involves the parenchymal cells (hepatocytes) almost exclusively. DNA synthesis usually begins at 15-18 hr and reaches a peak level at 24-26 hr. 9 Bile duct and supportive tissue cells enter DNA synthesis and mitosis much later; a maximal rate of DNA formation is not achieved until 36-42 hr posthepatectomy. Livers of younger animals (newborn or weanling rats) are restored more rapidly than in the adult animal. 9 In addition, a second wave of DNA activity occurs in weanling rats at 33-35 hr and represents proliferation of both parenchymal and nonparenchymal cells. 9 This probably indicates a relatively greater stimulation in weanling animals in which hepatic regeneration is superimposed upon an already rapidly
GROSFELD, WEBER, AND BAEHNER
growing liver. Under these conditions, presumably a larger portion of cells would have to divide repetitively during the first few days to compensate for the regenerative demand as well as to continue normal growth. These experimental findings are supported by the clinical observations in young children who have undergone partial hepatectomy. Sequential liver scans demonstrate rather rapid liver regeneration; postoperative metabolic parenteral support (adjunctive glucose, albumin, vitamin-K) is required for only a few days. Partial hepatectomy results in marked changes in the behavior of tbymidine metabolism. The activity of the synthetic pathway (incorporation to DNA) increases while the activity of the catabolic pathway (degradation to CO2) decreases. 6 Therefore, study of these opposing pathways are very useful to evaluate hepatic differentiation, regeneration and the effect of antimetabolites on the liver cell. In addition, these metabolic parameters are useful in studying the cellular metabolism of tumors. 6 Murthy et al. demonstrated that the nucleic acid and protein content of liver tumors in children are related to nucleocytoplasmic ratios and histologic maturity of the tumor. ~~ They noted that the DNA content of hepatoblastomas exceeded those of control livers. However, DNA content of hepatocarcinoma was similar to that of control liver content. Ferdinandes et al. 6 observed that incorporating thymidine 2-~4C into DNA increases and concurrently the degradation to CO2 decreases parallel with hepatoma growth in rats. The ratio of synthetic:catabolic pathways correlated directly with hepatoma growth. This imbalance was actually more pronounced than in the regenerating liver. They concluded that this marked imbalance in thymidine metabolism was linked with tumor growth but may also be related to a neoplastic alteration of gene expression. Labow et al. H theorized that the observed rise in enzyme synthesis following partial hepatectomy resulted from either a depression or induction of closely linked genes. These "forces" were considered capable of restoring the balance of the steady liver state after the wave of hepatic regeneration reached completion. This was signified by reestablishing the number of parenchymal cells and
CHEMOTHERAPY TOXICITY FOLLOWING HEPATECTOMY
953
restoring preoperative liver weight and liver:body ratio. This report confirms previous observations concerning the deleterious effects of Actinomycin-D on the regenerating liver. 3'5'6ActinomycinD combines with deoxyguanine residues to inhibit DNA-directed RNA synthesis. As expected, in rapidly proliferating liver cells, RNA mediated protein synthesis as well as nuclear division is impaired. Similarly, the anthracycline, Adriamycin, also impairs the synthetic capacities of cells in active cycle by blocking DNA biosynthesis by forming complexes directly with DNA. These data demonstrate that adriamycin also significantly inhibits DNA synthesis and interferes with liver regeneration. The lowest liver weights and least numbers of mitosis/HPF were observed using this drug. Previous data from our laboratory indicated that vincristine may have a protective effect on the intact liver exposed to the damaging effect of Actinomycin-D and irradiation and prevent the onset of radiation hepatitis. 5 In contrast to
Actinomycin-D and Adriamycin, Vincristine exerts its effect on cell division by binding to tubulin, the subunit structures required for microtubules to polymerize into spindle threads. This drug merely blocks mitosis but has no effect on DNA, RNA, or protein synthesis. Additional studies evaluating Vincristine in rats with 70% partial hepatectomy suggest that this drug does little to interfere with hepatic regeneration. 8 The present report confirms this latter finding. Following partial hepatectomy, Vincristine treated animals were similar to controls in regard to thymidine incorporation into DNA, liver weight and overall progress of regeneration. Our observations in this study indicate that Adriamycin and Actinomycin-D should be withheld until liver regeneration is near completion. The data also suggest that Vincristine may prove useful in the early posthepatectomy state when there is suspected microscopic residual disease or incomplete resection since it has a less injurious effect on regenerating residual liver tissue.
REFERENCES
1. Exelby PR Filler RM, and Grosfeld JL: Liver tumors in children in the particular reference to hepatoblastoma and hepatocellular carcinoma. J Pediatr Surg 10:329-337, 1975 2. Clatworthy HW, Schiller M, Grosfeld JL: Primary liver tumors in infancy and childhood: 41 cases variously treated. Arch Surg 109:143-147, 1974 3. Filler RM, Tefft M, Vawter GF, et al: Hepatic lobectomy in childhood: Effect of x-ray and chemotherapy. J Pediatr Surg 4:31-41, 1969 4. Ingold JA, Reed GB, Kaplan HS, et al: Radiation hepatitis. Am J Roentgenol 93:200-208, 1965 5. Grosfeld JL, Cooney DR, Shidnia H, et al: The effect of combined cancer therapy on the intact liver. Surg Forum 26:426-428, 1975 6. Ferdinandus JA, Morris HP, Weber G: Behavior of opposing pathways of thymidine utilization in differentiat-
ing, regenerating and neoplastic liver. Cancer Res 31:550556, 1971 7. Samuels LD, Grosfeld JL, Kartha M: Radiation hepatitis in children. J Pediatr 78:68 73, 1971 8. Grosfeld JL, Ferdinandus L, Cakmak O, et al: Safe use of Vincristine following partial hepatectomy. Surg Forum 27:89-91, 1976 9. Bucher NLR, Swaffield MN, Ditroia JF: The influence of age upon the incorporation of thymidine 2-~4C into DNA of regenerating rat liver. Cancer Res 24:509-512, 1964 10. Murthy ASK, Vawter GF, Kipito L, et al~ Biochemical studies on liver tumors of children. Arch Pathol 96:4852, 1973 11. Labow R, Maley GF, Maley F: The effect of methotrexate on enzymes induced following partial hepatectomy. Cancer Res 29:366-372, 1969
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GROSFELD, WEBER, AND BAEHNER
Discussion Daniel M. Hays (Los Angeles): Five years ago we were not sure if adjuvant chemotherapy played any role in managing patients with complete hepatic tumor resection. Today I think we can say that adjuvant therapy does clearly play a role. The current CCSG study demonstrates that patients with either hepatoblastoma and hepatocarcinoma have a 2-yr survival rate of 90% when the tumor is completely excised if they receive a four-drug chemotherapy regimen for 1 yr. Dr. Grosfeld's study is of particular importance because all of the chemotherapeutic systems should work more effectively early in the postexcision interval. The aim is to try and find the earliest time at which the chemotherapy can be started. I like Dr. Grosfeld's concept that liver regeneration need not be complete before starting chemotherapy. I have calculated the vincristine dosage as three times the amount that one would give in human medicine; the dactinomycin as about two times the human dose; and the adriamycin as about equal to the human dose, so he has given sufficient vincristine. I do not think one should infer that adriamycin is less hepatotoxic than actinomycin D; it is probably more
others (notably in this study, Vincristine) do not cause these problems. The clinical implications of this study are quite clear. We must know the action of each chemical agent on hepatic regeneration before its use in close proximity to surgery. The same applies to radiation therapy. We have previously reported studies with radiation therapy and Actinomycin D and their effect on hepatic regeneration. An additional aspect of this problem worthy of consideration is that these chemotherapeutic agents are ordinarily detoxified in the liver; therefore, when hepatic resection is performed, detoxification is impaired. Safe doses when the liver is intact may prove to be toxic after hepatecomy. Thus, the drug not only limits regeneration, but resection limits drug detoxification. One question I would ask Dr. Grosfeld is whether he has any information about the effect of these agents before hepatic resection? We are all familiar with recent reports about preoperative chemotherapy in children with hepatic tumors for "inoperable" tumors. I wonder if this approach might also limit liver regeneration.
SO.
Jay Grosfeld (Closure): I think Dr. Filler's question certainly is of interest. Actually, there is some correlary between hepatoma growth and the D N A synthetic pathway. The D N A synthetic rate of hepatoblastoma is similar to the regenerating liver and suggests that this tumor should be very susceptible to preoperative chemotherapy. This observation lends credence to Dr. Vos' comments regarding shrinking a large tumor in selected cases. Evaluating thymidine metabolism in the liver tumors may yield a prognostic factor. If the tumor has the ability to incorporate D N A it should be more responsive to chemotherapy. We plan to also study the effect of other drugs such as Bleomycin, Cytoxan and 5FU, (currently being employed in patients with liver tumors) on thymidine metabolism. Dr. Hays' comments were very kind. There were no statistical differences observed between adriamycin and actinomycin so what he said is absolutely correct.
Anton Vos (Netherlands): At the Children's Hospital in Amsterdam we recently have adopted a regimen using preoperative chemotherapy. We have treated three children with angiographic evidence of both liver lobes involved with hepatoblastoma. After several courses of chemotherapy (in one case over a total period of 10 mo) repeat angiography showed that the tumor was restricted to one lobe, which was subsequently resected. All three children are 2 yr postoperation, are free of disease and do not receive any chemotherapy. What Dr. Hays was talking about is the regeneration after the operation. Our experience is that on the liver scan made 6 mo after operation, the size of the liver had returned to normal. R.M. Filler (Toronto): This study gives both biochemical and morphological evidence that certain chemotherapeutic agents inhibit liver regeneration after hepatic resection, whereas