Burrell, N

Furuya, T

Iwatsuki, S

Matsuzaki, T

Murase, N

Shimizu, Y

Shinozuka, H

Starzl, TE

Yagihashi, A

English

PubMed

Name not available

Liver transplantation for diethylnitrosamine-induced hepatocellular carcinoma in rats

D-Scholarship@Pitt

/~ijl/ Liver Transplantation for Diethylnitrosamine-Induced Hepatocellular Carcinoma in Rats T. Matsuzaki, N. Murase, A. Yagihashi, H. Shinozuka, Y. Shimizu, T. Furuya, N. Burrell, S. Iwatsuki, and T.E. Starzl H UMAN hepatocellular carcinoma (HCC) is a highly malignant tumor with a natural history of a few months from the time of diagnosis. Curative resection offers the best chance of survival. l However, surgical resectability rates rarely exceed 30% to 40% because of extensiveness of the tumors or because coexisting liver disease precludes attempted partial hepatectomy.2 Conse-quently, total liver removal and replacement have been used extensively as a therapeutic option. 3 Because the rate of recurrence after liver transplantation has been high,3-9 it has been speculated that the requisite immunosuppressive agents may enhance the recurrence of original malignancies by weakening immune surveil-lance.3- lo In the present study, we have tested this hypoth-esis by inducing HCC in rats by multiple injections of diethylnitrosamine (DEN), followed by isogeneic liver transplantation with and without immunosuppression with FK 506 and cyclosporine (CyA). The results were incon-clusive. MATERIALS AND METHODS Animals and Induction of HCC Five- to 6-week-old male Fisher rats (F334) weighing 1 IO to 120 g were obtained from Harlan Sprague-Dawley, Inc (Indianapolis, IN) and given 75 mg/kg weekly intraperitoneal injections for 3 weeks of DEN (Sigma Chemical Co, St Louis, MO). followed by three further weekly injections of 100 mg/kg. The animals were maintained in a special room for carcinogen use with water and regular rat chow ad libitum. Isogeneic Fisher rats weighing 200 to 300 g were kept in a separate room until they were used as liver donors. Immunologic Testing Effector Cells. Mononuclear cells were isolated from the liver and spleen of normal Fisher rats and rats with HCC by morcella-tion of the specimen. and digestion for 15 to 20 minutes in RPMI medium (Gibco. Ground Island, NY), which was supplemented with 0.05% collagenase (Type 4, Sigma) and 0.002% DNase (Type I, Sigma). After passage through nylon mesh, the mononuclear cells were separated by Ficoll-Hypaque gradient. In the rats with HCC, bits of the liver tumor were processed from which the tumor infiltrating lymphocytes (TIL) were purified. TarRet Cells. The ability of the purified elfector cells to lyse three kinds of cell targets was determined: (I) Y AC-I line for natural killer (NK) cell activity, (2) P8151ine for lectin-dependent cell cytotoxicity (LDCC) in the presence of 5 J.tg/mL of phytohe-magglutinin P (Sigma). and (3) HCC cells isolated from a tumor in the same rat providing the TIL. The HCC cells were separated after enzyme digestion and discontinuous (75/100%) Ficoll-Hy-paque centrifugation (upper interface). 748 Target cells were placed in 96-well V -bottom plates (Coster. Cambridge, MA). and incubated with effector cells at effector/ target ratios ranging from 50: I to 6: I. Miniaturized 4-hour 51Cr_ release assays in triplicate were performed using I x 103 target cells. labeled with 51Cr (5 mCi/mL: New England Nuclear, Boston, MAL Maximal and spontaneous release were determined by adding 5% Triton X-IOO or medium. respectively. The plates were centrifuged at 651{ for 3 minutes and incubated at 37°C in 5% CO2 in air for 4 hours. Supernatants were harvested and radioac-tivity was counted with a beta scintillation counter. The percent specific lysis was determined according to the formula: % specific lysis = experimental release - spontaneous release -----------------------------x 100 maximal release - spontaneous release Lytic units of cytotoxicity were calculated according to the formula of Pross et al": one lytic unit was defined as the number of effector cells needed to lyse 20% of 5 x 10' target cells and cytotoxicity was calculated per 1 07 effector cells. Immunosuppressive Agents Oral FK 506 (Fujisawa Pharmaceutical Co Ltd. Osaka, Japan) dispersed with hydroxy propyl methylcellulose (a water-soluble polymer), was suspended in water with sonication and used at an oral dose of 1.0 mg/kg/d. Oral CyA (Sandoz Ltd. Basel. Switzer-land) was diluted with olive oil and used at a dose of 10 mg/kg/d. Drug administration was started I day after transplantation and continued for 150 days by direct instillation with a gastric tube. These doses of the two drugs are effective in preventing autoim-mune diabetes in rats 12.13 and in transplantation. OrthotopiC Liver Transplantation Orthotopic liver transplantation was performed with a modifica-tion of the method of Kamada and Calne,14 using the simplified cuff technique for the portal vein and infrahepatic vena cava anastomosis. Hepatic arterial reconstruction was not performed. Recipient animals received lM injection of cefamandole nafate (20 mg/d) for 3 days po,toperalively. From the Departments of Surgery (T.M., N.M .. T.F., N.B., S.I., T.E.S.), Pathology (A.Y., H.S.). and Pittsburgh Cancer Institute (Y.S.), University Health Center of Pittsburgh, University of Pitts-burgh. and the Veterans Administration Medical Center of Pitts-burgh, Pittsburgh. Pennsylvania. Supported by research grants from the Veterans Administration and proiect grant no. DK 29961 from the National Institutes of Health, Bethesda, Maryland. Address reprint requests to Thomas E. Starzl, MD, PhD, De-partment of Surgery, 3601 Fifth Ave, University of Pittsburgh. Pittsburgh, PA 15213. OJ 1992 by Appleton & Lange 0041-1345/92/$3.001+0 Transplantation Proceedings, Vol 24, No 2 (April), 1992: pp 748--751 HEPATOCELLULAR CARCINOMA 749 Table 1. Unaltered HCC Development by Histopathology, HCC Size, and Blood Chemistries in 24 Killed Animals Blood Chemistries Weeks After Mean HCC Total Last DEN Histopathologic Lung Size (cm) GOT GPT Bilirubin AKP GGT Albumin Injection n Diagnosis Metastasis (Range) (U/L) (U/L) (mg/dL) (U/L) (U/L) (g/dL) 5-7 9 GST-P positive foci 187 ± 65 96 ± 33 0.05 ± 0.05 176 ± 126 62 ± 40 3.74 ± 0.75 9-13 7 GST -P positive foci 0.26 122 ± 52 65 ± 35 0.02 ± 0.04 184 ± 26 33 ± 29 3.76 ± 0.42 and nodule 4 of (0.1-0.5) 7HCC 18-25 4 HCC with vascular 1/4 1.7 347 ± 173 335 ± 219 0.08 ± 0.05 231 ± 56 90 ± 48 4.13 ± 0.26 invasion (0.5-2.0) 32-40 4 HCC with vascular 2/4 2.15 831 ± 1,122 690 ± 880 0.25 ± 0.13 318 ± 126 291 ± 205 3.73 ± 0.70 invasion (0.6-5.0) Note: Values are mean ± SD Abbreviations: GST-P, glutathione S transferase placental form; GOT, glutamie-oxaloacetie transaminase; GPT. glutamic-pyruvic transaminase; AKP, alkaline phosphatase; GGT, gamma-glutamyl transpeptitase. Experimental Design Natural History. A total of 99 rats were entered. of which 6 were discarded 5 to 22 weeks after completion of the DEN course because of their morbid condition. Of the 93 remaining rats. 24 were followed and killed in cohorts to establish the natural history of the induced disease (Table 1). Liver and lung tissues were fixed in Stieve solution or Bouin's solution and paraffin embedded. Sections were stained for hematoxylin and eosin (H&E) and for glutathione S transferase placental form (GST-P), which is a marker of preneoplastic and neoplastic lesions of rat liver. 1.' Staining was with the avidin-biotin complex (ABC) method using antirat GST-P antibody (Bio Prep, Dublin. Ireland). Experimental Groups. All the other 69 rats developed HCC and were stratified by tumor size into experimental groups. Beginning III weeks after completion of DEN. laparotomy was performed. Rats with definite HCC nodules < 1.0 cm or with nodules> 1.2 cm were submitted immediately to orthotopic liver transplantation or consigned to a nontransplant group. The remaining rats with undetectable or small tumors were closed and periodically reop-erated every 3 to 4 weeks until the liver tumors appeared and qualified the animal for transplantation by reaching a size > 1.2 cm. A total of 42 laparotomies and 56 transplantations were performed on the 69 animals between 10 and 25 weeks after the last injection of DEN. Eighteen of 56 transplanted animals died within 7 days after surgery, and were excluded from further analysis. Posttransplant survival rate (67.9%) was poor when compared with those of other studies at our laboratory in which normal healthy rats were used. 16 The rats were placed into the groups summarized in Table 2. Except for the animals with small HCC (group 2) that all under-Table 2. Experimental Groups HCC Size OLTX at Entry* Immunosuppression Group N (Isograft) (em) AfterOLTX 13 >1.2 2 10 + <1.0 3 11 + >1.2 4 11 + >1.2 FK 506 1.0 mg/kg/d (po) 5 16 ~ >1.2 CyA 10.0 mg/kg/d (po) Abbreviations: OLTX, orthotopic liver transplantation. *Entry: See text for time of entry. went orthotopic liver transplantation I II weeks after completing DEN, the time of orthotopic liver transplantation was evenly distributed between 15 and 25 weeks post-DEN. The primary end point was duration of survival after transplantation. Statistical AnalYSis Differences between groups were analyzed by two-tailed Stu-dent's t test with the level of significance at P < .05. RESULTS Natural History In the animals killed 5 to 13 weeks after completion of DEN. 4 of 16 had frank HCC and all of the others had hyperplastic nodules or foci with GST-P positive stain that identifies premalignant changes (Table 1). Between 18 and 40 weeks, eight of eight animals had frank HCC and in three there were pulmonary metastases. Although only a few animals became jaundiced or had hypoalbuminemia. the transaminases and alkaline phosphatase were progres-sively increased (Table I). Effector Cell Activity The cytotoxicity of N K cells from the liver and spleen of HCC bearing rats was significantly reduced from that in normal animals (Table 3). The cytotoxicity of NK cells from the tumor (TIL) was even more drastically reduced. Cytotoxicity against autologous tumor cells was nil. HCC status did not affect the cytotoxicity against lectin-depen-dent target cells (Table 3). Effect of Transplantation and Immunosuppression The animals not submitted to either isotransplantation or immunosuppression lived for a median of 62 days (30 to ISO); two had pUlmonary metastases (Table 4). Survival was almost doubled by isogeneic transplantation. both in animals with small HCCs (group 2) and those with tumors > 1.2 cm (group 3). The tumors did not recur in any of the transplanted livers in groups 2 and 3, although pulmonary metastases were present at the time of death in 3 of the II rats of group 3. 750 MATSUZAKI, MURASE, YAGIHASI ET AL Table 3. Cytotoxicity of Mononuclear Cells from HCC-Bearing and Normal Rats Against Variable Target Cells Target Cells Autologous YAC·t P815 (-) pa15 (+) HCC Cells HCC-bearing rat (n = 3) TIL 16 ± 4' 0 0 0 Liver 55 ± lOt 1 ± 1 26 ± 5 0 Spleen 196 ± 32t 2 ± 1 26 ± 6 0 Normal rat (n = 3) Liver 246 ± 52 1 ± 1 10 ± 5 ND Spleen 337 ± 43 2±2 31 ± 8 ND Note: Cytotoxicities are shown in mean ± SO (LU20110' effector cells). Abbreviations: NO, Not determined .• P < .01 vs liver and spleen in HCC-bearing rats. t P < .05 vs liver and spleen in normal rats. The augmented survival obtained with liver replacement alone was reduced to a median of 18 days with FK 506 and to 54 days with CyA. Recurrent tumor in the transplanted liver of the immunosuppressed rats never was seen. and there were no examples of pulmonary metastases in the 15 experiments of groups 4 and 5. However, two rats devel-oped squamous cell carcinoma of the lung (in group 4) and another (in group 2) had developed a renal cell carcinoma. Surgical complications with the liver graft that did not have an arterial blood supply were common in all of the transplantation groups, and especially in those that in-cluded immunosuppression. Partial infarction of the livers with subsequent abscess formation was better tolerated in the nonimmunosuppressed animals than in those given FK 506 or Cy A. The death of animals with infarcted liver usually was due to overwhelming intra-abdominal sepsis as well as lung infection. This was particularly evident in rats under immunosuppression. DISCUSSION As expected. these studies showed that isogeneic hepatic transplantation could increase survival of rats bearing carcinogen-induced HCCs. It has been argued that this experimental HCC may not be relevant to human HCC because of the large amount of carcinogen administered and because the immunogenicity of the resulting tumors is high.17 These objections were answered in part by our demonstration that the general N K cell activity was de-pressed in these animals by the presence of HCC. Further-more. the tumor cells elicited little if any cytotoxic re-sponse (autologous) from the mononuclear cells of the tumor-bearing animals. Similar depressed immunologic reactivity is typical in humans with HCC. IS . 19 and has prompted a hypothesis that factors produced by human HCC cell lines are responsible. 20 Further exploration of the host-tumor relationships with this induced tumor should be fruitful but with the impor-tant modification that the liver grafts should be arterial-ized. The liver transplantation technique of Kamada and Calne l4 that provides good results in our hands when used in healthy animals 16 was not so well tolerated in the rat recipients already debilitated by DEN pretreatment. In these rats, the portal hemodynamics may have been al-tered by the DEN-induced liver disease. including a reduc-tion in portal venous flow and increased dependence on the hepatic artery as occurs in humans with cirrhosis. There was an extraordinary incidence of regional hepatic infarction that caused a high mortality in all animals, but Table 4. Animal Survival After Liver Transplantation With/Without Immunosuppression HCC (Primary/ Reeu rrence) Mean Time Complications %of Ani-Mean From Last DEN % of Animals mals HCC Size at Injection to Entry' (cm) Entry (d) Median Abscess in Group N (Range) (Range) Survival aiter Entry (d) Survival (d) Liver Liver 13 2.0 148.8 30,37,40,41,44,58,62,75,85,112,123,128,150 62.0 0 100 (1.2-3.2) (114-173) (13/13) 2 10 0.8 82.2 8,15,19,33,90,127,157, >196. >196, >205 108.5 40 0 (0.5-1.0) (66-82) (4/10) 3 11 1.6 106.3 8,18,17,15,62,101,113,126, >164, >164, >171 101.0 36.4 0 (1.3-2.0) (103-158) (4/11 ) 4 11 1.6 125.6 7,8,11,17,18,18,19,70,122,129, >164 18.0 63.6 0 (1.2-2.0) (107-166) (7/11 ) 5 6 1.9 144.6 ~ 12, 1~ 94,118, >154 54.0 50 0 (1.5-3.0) (124-173) (3/6) 'Entry: See Table 2. Time of entry for group 1, HCC size >1.2 em and no OLTX; group 2, HCC size <1.0 cm and OLTX; groups 3-5, HCC size "1.2 em and OLTX. HEPATOCELLULAR CARCINOMA especially those under immunosuppression whose death almost always was due to hepatic or hepatic plus pulmo-nary sepsis. Thus, before it can be determined if hepatic tumor recurrence or extrahepatic metastases are accelerated ei-ther by transplantation itself or by immunosuppression, more consistent long-term survival with fully vascularized grafts will be a necessary condition. Such a study in rats is more apt to resolve these questions than attempts to study human cases, which of necessity are uncontrollably heter-ogeneous.9 With the DEN method of HCC induction, the lesion being examined is producible in essentially 100% of animals, and its evolution can be determined under a wide range of controlled therapeutic variables including trans-plantation. REFERENCES 1. Nagasue N, Yukaya H, Hamada T, et al: Cancer 54:1461, 1984 2. Bismuth H, Houssin D, Omowski J, et al: World J Surg 10:311, 1986 3. Starzl TE, Putnam CW: Experience in Hepatic Transplan-tation. Philadelphia: WB Saunders Company, 1969 4. Bismuth H, Castaing D, Ericzon BG, et al: Lancet 2:674, 1987 751 5. OttoG, Heuschen UA, Hofmann WJ, etal: Eur J SurgOncol 16:346, 1990 6. Ringe B, Wittekind C, Bechstein WO, et al: Ann Surg 209:88, 1989 7. O'Grady JG, Polson RJ, Rones K, et al: Ann Surg 207:373, 1988 8. Iwatsuki S, Gordon RD, Shaw BW, et al: Ann Surg 202:401, 1985 9. Iwatsuki S, Starzl TE, Sheahan DG, et al: Ann Surg (in press) 10. Starzl TE, Penn I, Putnam CW, et al: Transplant Rev 7: 112, 1971 11. Pross HF, Baines MT, Rubin P, et al: J Clin Immunol1:51, 1981 12. Murase N, Lieberman I, Nalesnik M, et al: Transplant Proc 23:551, 1991 13. Laupacis A, Stiller CR, Garden C, et aI: Lancet 1:10, 1983 14. Kamada N, Caine R: Transplantation 28:47, 1979 15. Sato K: Adv Cancer Res 52:205, 1989 16. Murase N, Kim DG, Todo S, et al: Transplantation 50:186, 1990 17. Scott OCA: Cancer Res 51:757, 1991 18. Moy PM, Holmes EC, Golub SH: Cancer Res 45:57, 1985 19. Miescher S, Whiteside TL, Carrel S, et al: J Immunol 136:1899,1986 20. Keong A, Herman J, Rabson AR: Cancer Immunol Immu-not her 15: 183, 1983 

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Liver transplantation for diethylnitrosamine-induced hepatocellular carcinoma in rats

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