Severe radiation-induced liver disease following ... - Wiley Online Library

Severe Radiation-Induced Liver Disease Following Localized Radiation Therapy for Biliopancreatic Carcinoma: Activation of Hepatic Stellate Cells as an Early Event CHRISTINE SEMPOUX,1 YVES HORSMANS,2 ANDRE´ GEUBEL,2 JOE¨LLE FRAIKIN,3 BERNARD E. VAN BEERS,4 JEAN-FRANC¸OIS GIGOT,5 JAN LERUT,5 AND JACQUES RAHIER1

Radiation-induced liver disease is recorded as a form of veno-occlusive disease. Its pathogenesis remains unclear even if the initial injury likely occurs in the endothelial cells of central veins. The aim of our study was to investigate liver morphological features in relation to a-isoform of smooth muscle actin expression in hepatic stellate cells in six patients treated by localized radiotherapy on the biliopancreatic area. Within the month after completion of treatment, an activation of hepatic stellate cells strictly confined to irradiated areas and coinciding with congestive changes was observed. At a later stage, collagen deposition gradually increased, replacing the congestive and destroyed areas. This new fibrotic tissue also contained numerous a-smooth muscle positive cells. Our data suggest that early hepatic stellate cells activation coinciding with congestive changes plays an important role in radiation liver injury and ensuing fibrosis. (HEPATOLOGY 1997;26:128-134.) Interest of clinicians, pathologists, radiologists, and radiation oncologists for radiation-induced liver disease (RILD) has been prompted by the report of severe diffuse hepatic lesions occurring after irradiation of at least 30 Gy for lung cancer, metastatic ovarian carcinoma, and lymphoma.1-3 Clinically and morphologically, RILD was recorded as a form of veno-occlusive disease whose evolution may vary from complete recovery to progression into a subacute phase preceding the development of a chronic fibrotic state with portal hypertension and liver failure.2-5 Pathogenesis remains unclear. However, the initial injury likely occurs in the endothelial cells of central veins and sinusoids.5,6 From a carcinologic point of view and because of the disappointing therapeutic results of low radiation doses given on the biliopancreatic region, improved delivery of higher doses given to more focalized areas has recently been proposed.7-9 Preliminary data suggest that this technique may indeed result in improved patient outcome.7,10-12 Abbreviations: RILD, radiation-induced liver disease; a-SMA, a-smooth muscle actin; HSC, hepatic stellate cell; MRI, magnetic resonance imaging; H&E, hematoxylin and eosin. From the 1Department of Pathology, 2Department of Hepato-gastroenterology, 3Department of Radiation Oncology, 4Department of Radiology, and 5Department of Digestive Surgery, University Hospital St-Luc, Louvain Medical School, Brussels, Belgium. Received September 12, 1996; accepted February 13, 1997. Address reprint requests to: Christine Sempoux, M.D., Department of Pathology, ANPS 1712, University Hospital St-Luc, Avenue Hippocrate 10, B-1200 Brussels, Belgium. Fax: 32-2-764-89-24. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2601-0018$3.00/0

The aim of this study was to investigate liver morphological features in relation to a-isoform of smooth muscle actin (aSMA) expression in hepatic stellate cells (HSC) in six patients treated by localized radiotherapy on the biliopancreatic area. PATIENTS AND METHODS Patients. The patient population included three patients with

Klatskin tumors, two patients with extra-hepatic bile ducts carcinoma, and one patient with pancreatic carcinoma. There were one woman and five men whose ages ranged between 35 to 71 years (mean: 55 years). Previous history of the patients was unremarkable except for one patient who showed an underlying chronic hepatitis C (case 4). Clinical presentation included jaundice and biochemical cholestasis in all cases. Two patients (cases 1 and 2) underwent preoperative localized external radiotherapy before liver transplantation. In case 1, the dose delivered on the tumoral area was 54 Gy. In case 2, the dose was 40 Gy and 5-Fluorouracil (5-Fu; 300mg/m2/d) was administered as adjuvant therapy during radiotherapy. Liver transplantation was performed after 1 and 2.5 months, respectively, after completion of radiotherapy. In the four remaining cases, radiotherapy was administered at lower doses than in the two first cases, as an adjuvant postoperative therapy after Whipple resection (cases 3 and 6) and/or left hepatectomy (cases 4, 5, and 6). In these four nontransplanted cases, postoperative external localized radiotherapy was associated with 5-Fu administration in one case (case 3; 300 mg/m2/d) and with local curietherapy with Iridium 192 in three cases (cases 4, 5, and 6). Local curietherapy was applied on the anastomotic site during 4 days at a dose of 20 Gy at a 5-mm depth. After completion of treatment, each patient underwent full clinical evaluation every 3 months together with biochemical work-up and magnetic resonance imaging (MRI). In the nontransplanted cases and because of the occurrence of ascites, jaundice, and abnormal liver function tests, hepatic venous catheterization and transvenous liver biopsy procedure was performed for case 3 at 2.5 months after a dose of 35 Gy; for case 4 at 4 months after a dose of 33 Gy, and for case 5 at 5.5 months after a dose of 32 Gy (Table 1). In these three patients, MRI performed at the time of histological examination showed a sharply delineated area of liver parenchyma with decreased signal intensity on T1weighted images and increased signal intensity on T2-weighted images. In the remaining patient, hepatic venous catheterization together with a transvenous liver biopsy was performed 8 months after radiation therapy with 12 Gy in the absence of any MRI abnormality because of the persistence of abnormal liver function tests (case 6). Clinical characteristics and evolution, therapeutic manipulations, biochemical parameters, and wedged hepatic vein pressure gradients observed in the reported cases are listed in Table 1. Histological Study. Liver biopsy specimens were fixed in Bouin’s solution for 24 hours whereas hepatectomy specimens were fixed in 4% buffered formaldehyde solution for 48 hours. The material was embedded in paraffin, cut in 6-mm thick sections and stained with hematoxylin and eosin (H&E), Masson’s trichrome for colla-

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TABLE 1. Clinical Characteristics and Evolution, Therapeutic Manipulations, Biochemical Parameters, and Wedged Hepatic Vein Pressure Gradients of the Reported Cases

Cases

Age at the Time of Surgery

Sex

Chemotherapy

Curietherapy

Radiotherapy

Delay Between the End of Treatment and the Liver Biopsy

1 2 3

58 50 66

M M M

— /5 FU /5 FU

— — —

54 Gy 40 Gy 35 Gy

1 mo 2.5 mo 2.5 mo

65/172/1.3

7

4

71

F

—

/

33 Gy

4 mo

101/181/1.31

7

5

50

M

—

/

32 Gy

5.5 mo

91/146/0.5

1

6

35

M

—

/

12 Gy

8 mo

182/58/0.43

1

Biological Results at the Time of Liver Biopsy ALT*/Alk. Ph†/ Bili‡

Wedged Hepatic Vein Pressure Gradients (N õ 4 mmHg)

Evolution

Orthotopic liver transplantation Orthotopic liver transplantation Ascites occurring 7 weeks after radiotherapy and persisting during 7 months Ascites occurring 8 weeks after radiotherapy and persisting during 4 months Ascites occurring 9 weeks after radiotherapy and persisting during 3 months Liver function tests disturbance

Outcome

Liver atrophy; death at 22 mo Tumoral recurrence at 15 months; death at 24 mo Well at 30 mo

Well at 34 mo

* ALT, Alanine aminotransferase (N õ 32 IU/L). † Alk.Ph., Alkaline phosphatase (N õ 60 IU/L). ‡ Bili., serum total bilirubin (N õ 1 mg/dL).

gen fibers, and Foot’s method for reticulin. a-SMA expression in HSC was investigated using a monoclonal antibody directed against the a-isoform of SMA (Dakopatts, Glostrup, Denmark) at the working dilution of 1:150 in a standardized immunohistochemical procedure with the streptavidin-peroxidase system.13 Slides were incubated overnight at room temperature and the peroxidase activity was revealed by immersion of the sections for 10 minutes in a solution of 3,3-diaminobenzidine hypochloride (50mg/100mL at pH 7.4; Amersham, Cardiff, UK), supplemented with 0.02% H2O2 . Vascular smooth muscle cell immunoreactivity was used as an internal positive control. RESULTS

In cases 1 and 2, total hepatectomy specimens were obtained, weighing respectively 1,606 g and 1,150 g. In irradiated areas, large congestive zones were observed, whereas nonirradiated parenchyma did not show any macroscopic abnormality. Microscopic examination of the irradiated areas revealed a striking sinusoidal congestion in the centrilobular and intermediate zones (Fig. 1A) resulting in compression together with atrophy of liver cell plates (Fig. 1B). The central veins were difficult to identify because of a variable thickening of their walls by densely packed erythrocytes (Fig. 1C) as well as by the deposition of reticulin fibers sometimes obliterating their lumen (Fig. 1D). Reticulin network was also considerably increased in sinusoids of the centrilobular area. In case 1, severe congestive lesions focally involving the entire lobule were observed close to the liver hilum around a white, dense, and hard area microscopically corresponding to a well-differentiated adenocarcinoma included in an important fibrous background. In case 2, there was an irregular white and firm area surrounding the right hepatic duct in which at microscopic examination, only nontumoral bile ducts were observed in the absence of residual malignant tissue. Foci of congestion were less numerous and large than those observed in case 1. They also mainly involved the central portion of the lobules and coincided with an atrophy of the liver cell plates and a partially fibrotic central vein. Using trichrome stain, some fibrotic enlargement of the portal tracts and weak periductular fibrosis were focally seen.

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In the irradiated area of both cases and preferentially in the congestive part of the lobules, large and intensely stained aSMA positive cells were observed (Fig. 2A). Some of them accumulated in the thickened wall of the central vein and were closely associated with reticulin deposits (Fig. 2B) whereas others formed a network with a stellate pattern surrounding cords of atrophic hepatocytes adjacent to centrilobular engorged sinusoids (Fig. 2C). The distribution of activated HSC was however uneven. Indeed, some congestive areas were devoided of any a-SMA positive cells or showed brightly stained cells in the periportal, fewer congestive area. Numerous positive HSC were also observed in the portal tracts and in the fibrous peritumoral stroma. On the contrary, nonirradiated parenchyma was devoided of any significant microscopic lesion and contained only rare a-SMA positive HSC with thin cytoplasmic processes scattered along sinusoids and showing only a light brown staining (Fig. 2D). In case 3, the biopsy specimen was characterized by the same congestive lesions as those observed in cases 1 and 2 together with a proliferation of brightly a-SMA positive HSC in enlarged sinusoids. In case 4, the biopsy showed a distortion of the lobular architecture with expanded fibrotic portal tracts, some degree of bile duct proliferation, and areas of centrilobular perisinusoidal fibrosis (Fig. 3A, B, and C). Some focal sinusoidal congestion was still present and several star-shaped a-SMA positive cells were irregularly distributed in fibrotic areas (Fig. 3D). In case 5, liver architecture was preserved with some portal and sinusoidal fibrosis. Moderate centrilobular steatosis with mild cholestasis and some necrotic cells were noted. Reticulin framework was focally slightly increased. Some rare thin a-SMA positive cytoplasmic processes were focally noted in sinusoids. In case 6, the biopsy specimen showed a preserved liver architecture without appreciable change of portal tracts or centrilobular regions. Hepatocytes showed moderate macrovacuolar steatosis. Trichrome staining showed a weak and focal sinusoidal fibrosis, whereas Foot’s method showed a

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FIG. 1. Typical morphological features of acute RILD observed in irradiated areas of case 1 hepatectomy specimen. (A) Severe congestion involving the central part of the lobules with preserved portal tracts showed by arrows (H&E; original magnification 1128). (B) Compression and atrophy of liver cell plates (H&E; original magnification 1520). (C) Sclerotic central vein with densely packed erythrocytes in the thickened venous wall (H&E; original magnification 1520). (D) Increased reticulin network in the wall of the central vein as well as in the surrounding parenchyma (Foot’s staining; original magnification 1520).

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FIG. 2. Immunohistochemical expression of a-SMA in acute RILD observed in case 1 hepatectomy specimen. (A - C) Irradiated areas. (A) Accumulation of numerous a-SMA positive HSC in the congestive parts of the lobules. In this picture, parts without congestion or liver cell necrosis show no HSC proliferation. (Original magnification 1128.) (B) High magnification of Fig. 2A focusing on the striking centrilobular accumulation of a-SMA positive HSC. The central vein is no longer visible being probably obliterated by this proliferation. (Original magnification 1320.) (C) Characteristic stellate morphology of a-SMA positive HSC along the distal ends of congestive sinusoids. (Original magnification 1520.) A few Kupffer cells containing hemosiderin, shown by arrows, are scattered between hepatocytes with lipofuscines in their cytoplasms. (D) Nonirradiated area. Only rare and weakly positive HSC are seen in the Disse’s space. (Original magnification 1520.)

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FIG. 3. Fibrotic phase of RILD observed in case 4 liver biopsy specimen. (A) Distortion of lobular architecture related to the presence of irregular fibrotic areas. (H&E; original magnification 1128.) (B) Higher magnification showing inflammatory cells and some ductular proliferation. (H&E; original magnification 1520.) (C) Densification of the reticulin network with architectural distortion. (Foot’s staining; original magnification 1320.) (D) Several a-SMA positive HSCs scattered in a fibrous periportal area. The portal tract is seen in the center of the picture, the short arrow showing the portal vein and the long arrow the bile duct. (Immunoperoxidase staining; original magnification 1520.)

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patchy densification of the reticulin framework. Thin cytoplasmic processes rarely and focally located along the sinusoidal walls were weakly positive for a-SMA. DISCUSSION

In this series and from a pathological point of view, the five cases of partially irradiated liver with doses ú30 Gy showed the whole range of morphological features seen after liver irradiation. Cases 1, 2, and 3 showed important sinusoidal congestion together with bleeding, atrophy, and necrosis of zone 3 hepatocytes related to phlebosclerotic lesions of the central veins, a picture of veno-occlusive disease well described in the acute and subacute phases of the disease.2-5 Some of the congestive lesions might have been the result of flow abnormalities adjacent to space occupying lesions. However, the preferential centrilobular localization of the congestive features together with the presence of sclerotic central veins, which are hallmarks of veno-occlusive disease, are strong arguments in favor of radiation-induced lesions in the first two cases. Moreover, in case 3, similar lesions appeared in the absence of any tumoral recurrence. The nonirradiated areas remained free of abnormality. In case 4, the chronic phase of RILD with large areas of fibrosis in lobules together with the persistence of some focal congestion was observed. In case 5, recovery stage of the disease was seen, with some cicatricial lesions, mainly consisting in some portal and sinusoidal fibrosis and focally in slightly abnormal reticulin network. Case 6, which received doses õ 30 Gy did not show any significant morphological lesion. From a clinical point of view, our observation indicates that partial liver irradiation with doses higher than 30 Gy may induce cholestasis, jaundice, ascites, and liver atrophy. In the acute phase these clinical features were found to be associated with congestion, bleeding, and destruction of liver cell plates and in the chronic phase, with fibrosis. From a radiological point of view, three patients of our series showed MR imaging evidence of a sharply demarcated liver region of lower signal intensity corresponding to the irradiation field, a peculiar feature which has been recently reported by others.9 In two patients, this picture progressively resolved while in the third patient, it was progressively replaced by an atrophy of the corresponding liver segments. Clinically and morphologically, RILD was recorded as a form of veno-occlusive disease whose evolution may vary from complete recovery to the progression into a subacute phase preceding the development of a chronic fibrotic state with portal hypertension and liver failure.2-5 The mechanism of such lesions remains poorly understood. In 1980, Fajardo and Colby described the presence of focal depositions of fibrin in the central veins of irradiated livers and postulated that injury mainly involved endothelial cells. This fibrin network was eventually replaced by collagen deposition resulting in fibrous occlusion.5 In 1987, Shulman et al. brought further arguments in favor of this hypothesis showing by immunohistochemical methods deposits of coagulants within the veno-occlusive lesions.6 Our morphological data favor the existence of an early activation of HSC beginning within a month after completion of treatment and coinciding with congestive changes. This event occurs in the acute phase of the disease, the accumulation of a-SMA positive HSC being strictly confined to irradiated areas and essentially in the congestive zones of the lobules, adjacent to atrophic cords of damaged hepatocytes, sometimes thickening the wall of

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the central vein and closely associated with reticulin deposits. At a later stage, deposition of collagen was gradually increased replacing the congestive and destroyed areas, the newly formed fibrotic tissue also containing numerous aSMA positive cells. After any type of injury to the liver, HSC proliferate in the affected sites and undergo a myofibroblastic transformation preceding the production of extracellular matrix and hepatic fibrosis.14-18 Immunohistochemistry using an antibody directed against a-SMA, an actin isoform typically found in smooth muscle cells, is a sensitive and reliable method that allows the identification of both normal and transformed HSC in human liver.19-22 a-SMA positive HSC, scattered along hepatic sinusoids, are rarely seen in normal adult liver22 as exemplified in the nonirradiated parenchyma of our cases. In the course of RILD, it is tempting to speculate that in the acute phase of the disease, an endothelial injury resulting in fibrin deposition, blood flow obstruction, and extensive congestion causing hepatocyte atrophy and necrosis is associated with an activation of HSC. In the following fibrotic phase, activated HSC produce reticulin and collagen fibers that progressively replace the congestive areas by newly synthesized fibrous tissue. Furthermore, as suggested by Fig. 2B, radiation-induced proliferation of HSC in central vein might be an early event in the development of the phlebosclerotic lesion that in turn may be responsible for ensuing sinusoids congestion and liver cell atrophy. Activation of HSC is however probably only for a part responsible for sclerosis of the central vein because an association between HSC proliferation and phlebosclerotic lesion was not invariably observed. Different mechanisms may be potentially responsible for HSC activation. It may result from free-radicals release related to radiotherapy. It may also be secondary to congestion or to stimulating factors released by damaged hepatocytes, endothelial cells and/or Kupffer cells. Indeed, the fact that in the noncongestive and nonnecrotic parts of the irradiated liver lobules there was only limited HSC activation that favors the later hypothesis. In addition, numerous cytokines potentially produced by these cells, are known to modulate HSC activity.23-24 Among them, and to the best of our knowledge, the only one that has been found in the liver following exposure to external beam radiation is transforming growth factor-b1.25 As numerous studies have established a relation between transforming growth factor-b1 and HSC,26-31 it is tempting to speculate that the activation of HSC responsible for ensuing fibrogenesis is, at least partly, related to TGF-b1 release. In our series and from a clinical point of view, no pathological manifestation was observed after partial irradiation with doses õ 30 Gy. In contrast, the administration of doses in the 30-Gy range or higher induced cholestasis, jaundice, ascites, and liver failure within weeks or months following radiotherapy (Table 1). This observation suggests that partial liver irradiation may result in similar consequences to those observed after whole liver irradiation. In the weeks following radiotherapy, ascites occurred in three nontransplanted cases (cases 3, 4, and 5) and disappeared thereafter in all cases. This transient phenomenon was likely not related to the occurrence of portal hypertension of the postsinusoidal type because a main portion of the liver parenchyma remained unaltered and also because the wedged hepatic vein pressure gradient was normal or only slightly increased. Ascites production might thus mainly result from

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direct injury to lymphatic channels located into the liver hilum and/or into the irradiated field. It must also be outlined that no patient presented with the acute clinical picture of RILD and that patients who developed the subacute and chronic illnesses underwent other treatments that may have played an additive role to the damaging effect of radiotherapy. Such factors and especially chemotherapy have indeed been suspected to play a modulating effect in radiotherapy-induced liver damage.32-35 In conclusion, this study of a-SMA expression indicates that in the development of RILD, the activation of HSC limited to the irradiated area is an early event preceding fibrogenesis. Our observation also shows that doses higher than 30 Gy administered on a limited area may lead to severe clinical manifestations undistinguishable from those seen after total liver irradiation. However, the threshold value of 30 Gy may represent a great oversimplification and the role and importance of other modulating factors of injury remain to be elucidated. Acknowledgment: We thank S. Lagasse for phototechnical assistance. REFERENCES

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