668 @ 1991 The Japanese Society of Pathology

Hepatic Cytomegalovirus Involvement in Autopsy Cases

Nobuya Sano and Keisuke lzumi Hepatic involvement of cytomegalovirus (CMV) was studied in 15 autopsy cases with disseminated or solitary CMV infection. Formalin-fixed, paraffin-embedded tissue sections were examined by immunohistochemistry (IHC) using a monoclonal antibody against early and late CMV antigens, and by i n situ hybridization (ISH) using biotinylated CMV DNA probes. Three cases showed cytomegalic cells in liver sections by conventional staining, five showed hybridization with CMV DNA probes and seven showed reaction with the monoclonal antibody in the liver. CMV infection was detected not only in cytomegalic cells but also in many non-cytomegalic cells by IHC and ISH, proving these techniques to be superior to routine histological examination. The inflammatory reaction in the liver was not prominent. The reason for the weak inflammatory response in the liver of our present cases, and the possible availability of IHC and ISH for analysis of liver biopsy and bronchoalveolar lavage specimens from lmmunocompromised hosts were discussed. Acta Pathol Jpn 41 : 668-672,1991.

Japan. Recently, immunohistochernical and genetic procedures have been employed to detect CMV infection in formalin-fixed, paraffin-embedded tissue sections (1015). Furthermore, the significance of these procedures as rapid, sensitive and specific diagnostic tools has been emphasized. With the advent of the antiviral agent ganciclovir (9-[ 1, 3-dehydroxy-2-propoxymethyI] guanine), it has become increasingly important to identify patients with invasive CMV infection early in their disease course. The present study was performed: 1) to determine the value of routine histology, in situ hybridization (ISH) and immunohistochemistry (IHC), for detection of CMV infection, and 2) to investigate hepatic involvement of CMV in Japanese patients.

MATERIALS AND METHODS Key words: Cytomegalovirus, lmmunohistochemistry, In situ hybridization, Liver

INTRODUCTION Cytomegalovirus (CMV) is one of the most common opportunistic infectious agents in immunocompromised hosts, particularly bone marrow and organ transplant recipients (l),the patients with malignant disease receiving aggressive chemotherapy (1, 2) and also those with AIDS (1, 3). Although CMV infection has often been studied by conventional histological observation (4-9), the precise histological features of hepatic involvement by CMV have rarely been documented, particularly in ~~

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Received March 1, 1 9 9 c Accepted for publication June 10, 1991. Second Department of Pathology, The University of Toku shima, School of Medicine, Tokushima. Mailing address : Nobuya Sano, Second Department of Pathology, The University of Tokushima, School of Medicine, Kuramoto 3-18-15, Tokushima 770, Japan.

Fifteen autopsy cases, which had CMV inclusion bodies in one or more organs, were retrieved from files of 4 7 4 cases between 1 9 8 3 and 1988 at the Department of Pathology, The University of Tokushima, School of Medicine, To kushima, Japan. Formalin-fixed, paraffin-embedded liver tissues were cut into 4 p m sections for routine histology, immunohistochemistry (IHC) and in situ hybridization (ISH). A t least two blocks per case were used for the study. On routine histological examination, CMV-infected cells were identified using three criteria : 1) presence of an amphophilic nuclear inclusion surrounded by a clear space, 2) acidophilic to amphophilic cytoplasmic inclusions, and 3) cellular enlargement. A cell was deemed diagnostically cytomegalic if it fulfilled all three criteria, the first criterion only, or the second and third criteria together. For IHC, a commercially available monoclonal antibody, reactive with both early and late CMV antigens, was purchased from DAKOPATTS (M-757, DAKO,

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Copenhagen, Denmark). The peroxidase-labefed strep tavidin-biotin technique (Histofine SAB-PO kit, Nichirei Co., Tokyo, Japan) was performed using this monoclonal antibody. Tissue sections were dewaxed, rehydrated, depleted of endogenous peroxidase with hydrogen peroxide-methanol, and digested with proteinase K (0.01%, P0390, Sigma Chemical Co., St. Louis, MO, USA) a t 37°C for 3 0 min. Any non-specific reaction was blocked with rabbit serum, and the sections were incubated for 1 h at room temperature with anti-CMV monoclonal antibody diluted 1 : 50. Then the tissue sections were treated with biotinylated rabbit anti-mouse immunoglobulin, followed by streptavidin-horseradish peroxidase complex. A CMV DNA probe assay kit (PATHOGENE, Enzo Biochem, Inc., NY, USA) was used for ISH. Tissue sections were treated with hydrogen peroxide and proteinase K by the same method as that for IHC. Subsequently, the DNA probe was applied to the tissues, and the slides were placed on a heated block at 95°C for 5 min. The slides were then transferred to a moist chamber and incubated at 37°C for 1 h, and the tissue was covered with post-hybridization solution a t room temperature for 10 min. After rinsing in wash buffer, the slides were treated with avidin-peroxidase solution for 3 0 min. For IHC and ISH, the tissues were counterstained with hematoxylin after the chromogen reaction using diaminobenzidine and hydrogen peroxide. For positive controls, lung tissues with numerous cytomegalic inclusions were employed and for negative controls uninfected materials were used. For a comparison of the sensitivity of ISH and IHC, the number of positive cells per cmz was measured by

morphometry (Color Image Analyzer, Olympus Co., Tokyo, Japan). In the same manner, the number of inclusion bodies in the lungs was calculated in each case.

RESULTS Clinical and autopsy profiles and data obtained in this study are summarized in Table 1. The 15 patients comprised eight females and seven males, ranging in age from 3 2 to 7 2 years, with a n average age of 5 5 years. Hematopoietic malignancy was the most frequent underlying disease, followed by lung cancer. Of these 15cases, three cases (20%) showed CMVinfected cells in liver sections stained with HE. The lungs were the organs most often involved (loo%), followed by the adrenal glands (67%), by routine histological observation. Besides pulmonary, adrenal and hepatic involvement, six cases had CMV inclusions in other organs such as the digestive tract, pancreas, thyroid, kidneys, ovaries, spleen, lymph nodes and skeletal muscles. In these six patients with disseminated CMV infection, five had hepatic involvement detected by either of the three methods employed (cases 1-3, 5 and 7). In addition to CMV infection, six patients (cases 1-3, 5, 6, and 8 ) showed concurrent opportunistic infection with other agents. All of these six patients had moniliasis, and three (cases 1-3) were infected additionally with Aspergillus or herpes simplex virus. In five of these six patients, hepatic CMV infection was confirmed at least by IHC. Cytomegalic change was most frequently seen in hepatocytes among all infected cells in the liver in HE sections. Bile duct epithelial involvement was rare,

Table 1. Clinical and Infectious Profiles and Number of Infected Cells in the Liver and Lungs of Patients with CMV Disease Case

Primary disease

Patient Age I”_

__

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

a

Interstitial pneumonia SLE Brain tumor (SCC) Hodgkin’s disease AML Multiple myeloma Lung cancer Cholangiocarcinoma Malignant lymphoma Malignant lymphoma Takatsuki’s disease CMMoL Lung cancer Cervical cancer Skin tumor (SCCI

57 46 68 40 32 54 66 66 58 71 51 41 59 47 69

F F F M F M F M F M M F M F M

Concurrent opportunistic infection Candida, Aspergillus Candida, Aspergillus Candida, HSV

(-1

Candida Candida

(-1 Candida

(-1 (-1 (-1 (-1 (-1 (-1 (-I

Disseminate& CMV infection

+ + + + + -

-

-

-

+ -

No. of CMV infected cells/cm2 in liver by in lungs by HE ISH IHC HE

0.26 0.22 0.11 0 0 0 0 0 0 0 0 0 0 0 0

1.06 0.44 0.11 0.24 0.11 0 0 0 0 0 0 0 0 0 0

2.90 1.08 1.60 0.48 1.47 1.11 0.23 0 0 0 0 0 0 0 0

23.5 2.86 3.44 3.89 >lo00 0.36 0.93 9.35 2.65 24.4 2.12 7.11 0.29 0.80 0.88

Cytomegalic involvement other than lungs, adrenal glands and liver revealed by routine HE sections. SLE, systemic lupus erythematosus; SCC, squamous cell carcinoma ; AML, acute myelocytic leukemia ; CMMoL, chronic myelomonocytic leukemia ; HSV, herpes simplex virus.

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Cytornegalovirus Infection of the Liver (Sano and Izumi)

Figure 1. Inclusion-bearing hepatocyte in a cord without inflammatory reaction or necrotic change (HE).

being found only in case 1. There was no evident CMV infection in Kupffer cells and other sinusoidal cells. No preferential intralobular distribution of infected hepatocytes was found. Inflammatory cell accumulation around infected cells was an exceptional finding (Fig. 1). However, minimal patchy granulomas and acidophilic bodies were found in case 1. Diagnostic nuclear inclusions in hepatocytes were recognized by ISH with CMV DNA probe. Also, nuclear reactivity was evident occasionally even in normal, nonenlarged, liver cells. In some hepatocyteqthe cytoplasm was also labeled in the form of rough particles of varying size (Fig. 2a). IHC disclosed the same nuclear staining as that in ISH, and the cytoplasmic reaction was observed as fine uniform granules or as rough particles in some instances (Fig. 2b). In cases 1 to 7, the number of positive cells ranged from 0.11 to 0.26/cm2 by routine histology, from 0.11 to 1.06/cm2 by ISH and from 0.48 to 2.90/cm2 by IHC. Furthermore, infected cells were detected by IHC even in cases that were negative by routine histological observation or ISH. There was a discrepancy between the densities of infected ceHs in the lungs and those in the liver in some cases. Case5, where the patient had had the most severe CMV pneumonia, showed no hepatic CMV inclusions in routine sections. On the other hand, in case 6, which had the second lowest number of infected cells in the lungs among all 1 5 cases, the number of cells in the liver labeled by IHC was close to that of case5.

Figure 2. Positive cells lacking cytomegalic change. a : lntranuclear and intracytoplasmic staining for CMV DNA probes (ISH). b : Intense nuclear staining with monoclonal antibody against early and late CMV antigens. Mixture of fine granules and rough particles is evident (IHC).

DISCUSSION In reviewing autopsy and surgical materials, Henson et aL(16) found associated hepatitis in 11 (19%) of 5 9 cases of disseminated cytomegalic inclusion disease. Takayama (6) reported that inclusion bodies in the liver were found in 18% of Japanese autopsy cases with CMV infection. In our study, hepatic cytomegalic inclusion bodies were observed in three cases (20%) by conventional examination. With the aid of ISH and IHC, hepatic CMV infection was confirmed in additional four cases. Of these seven cases with hepatic CMV infection, five had disseminated organ involvement with CMV. In some cases, CMV infection results in generalized disease, usually diagnosed at autopsy in conjunction with other morphologically defined infections (2-9, 16). In our study, there were six cases with concurrent opportunistic infectious agent(s) other than CMV. Of these, five had hepatic CMV infection detected by routine histological observation, ISH or IHC. In our study, IHC was the most sensitive technique, followed in order by ISH and routine histology for detection of CMV. The higher sensitivity of IHC compared with ISH may be explained by the fact that CMV was detected at different stages of its replicative cycle by each technique. lmmunostaining with the monoclonal antibody against the early and late antigens may be able to detect cells in which viral replication has not yet occurred. These facts indicate that: 1) hepatic CMV involvement may be a sign of disseminated infection with the

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Acta Pathologica Japonica 41 (9): 1991

virus, 2) concurrent infection with other opportunistic agent(s) may suggest hepatic CMV involvement, and 3) immunohistochemical staining against the early and late viral antigens may detect CMV infection in its early stage. Several reports on hepatic CMV infection from western countries (7, 17, 18) have described findings of parenchymal necrosis with prominent inflammatory activity as “hepatitis”. In contrast, Takayama (6) reviewed Japanese autopsy cases and reported a low incidence of inflammatory infiltrates in the liver with CMV inclusion bodies. In our series, we could hardly recognize either inflammatory infiltrates around cytomegalic cells, or granuloma distributed randomly in the liver. We did not experience any cases which could have fallen into the category of “hepatitis” in the present study. Exceptionally, several granulomas and acidophilic bodies were found in case 1, but no inflammatory reaction was evident. These facts indicate that hepatocytic damage may be caused by the direct cytopathic effect of CMV rather than by immunologic attack against infected hepatocytes in Japanese patients, and that necrotic material may be removed after cell death by scavengers. However, the distribution and grade of inflammation may vary at different stages of the immunocompromised status. While most Japanese are vertically infected with CMV at delivery, a much lower proportion of the population in western countries has antibody against CMV in childhood(l9). The difference in the age of infection may contribute to the clinical and morphological variety of CMV hepatitis, as is the case in HBV infection. Demeteris et a/. (17) reported that in some cases of CMV infection, inflammatory activity in the liver became obvious with tapering of immunosuppressive therapy. Among immunocompromised hosts, CMV has gained most attention in the recipients of organ transplantation (1, 12, 14, 15, 17). In previous studies of CMV hepatitis, the diagnosis was made after detection of CMVinfected cells in the liver using histological (1-9, 16), immunological (10-15), genetic (10-15) or culture (12, 14, 15) methods on hepatic tissue. When the diagnosis was made, a large number of liver specimens might have shown an inflammatory reaction or destruction of hepatocytes prominent enough to be regarded as “hepatitis”. Conversely, our present study indicates that even minor hepatic CMV involvement may be a sign of systemic infection with the virus in Japanese patients. As shown in this study and others(2-9), the lung is one of the organs most often involved, and the liver is not a preferential site for CMV. Paya et a/. (20) reported that 30% of liver transplant recipients had CMV hepatitis and that 13 of 18 patients with invasive CMV infection had viremia regardless of the presence of hepa-

titis. These facts suggest that bronchoalveolar lavage combined with immuno histochemical examination (2 1, 22) may be one of the best approaches for detection of CMV infection a t the early stage, because of the pulmonary preference of CMV, low risk to immunocompromised hosts, high sensitivity and wider surveillance area compared with biopsy examination. In Japan, treatment of hepatic disease by transplantation has just begun. However, much still remains unknown about CMV infection, which is one of the most life-threatening factors for allograft recipients. Further studies will be required to clarify the pathogenesis of this common infectious agent in immunocompromised hosts and to obtain the most effective method of diagnosis and treatment. Acknowledgements : We are grateful to professor Hisashi Otsuka, the University of Tokushima, School of Medicine, for reviewing the manuscript and providing valuable comments.

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Cytomegalovirus Infection of the Liver (Sano and Izumi) histochemistry using new monoclonal antibody CCH2 : A comparison of methods. J Clin Pathol 41 : 10051009, 1988. Masih AS, Linder J, Shaw BW, et a/. Rapid identification of cytomegalovirus in liver allograft biopsies by in situ hybridization. Am J Surg Pathol 1 2 : 362367, 1988. Paya CV, Holly KE, Wiensner KE, et a/. Early diagnosis of cytomegalovirus hepatitis in liver transplant recipients. Hepatology 12 : 119-1 26, 1990. Henson DE, Grimley PM, and Strano AJ. Postnatal cytomegalovirus hepatitis. Hum Pathol 5 : 93-103, 1974. Demeteris D, Lasky S, Van Thiel DH, Starzl TE, and Dekker A. Pathology of hepatic transplantation. Am J Pathol 118: 151-161, 1985. Reller LB. Granulomatous hepatitis associated with acute cytomegalovirus infection. Lancet 1 : 20-22,

1973. 19. Numazaki Y. Cytomegalovirus infection in Japan. Nippon Rinsho 35: 2738-2742, 1977 (in Japanese). 20. Paya CV, Hermans PE, Washington JA Jr, et a/. Incidence, distribution and outcome of episodes of infection in 100 orthotopic liver transplantations. Mayo Clin Proc 64: 555-564, 1989. 21. Martin WJ Jr and Smith TF. Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by monoclonal antibody method. J Clin Microbiol 23 : 1006-1008, 1986. 22. Woods GL and Thiele GM. Rapid detection of cytomegalovirus by 24-well plate centrifugation with the use of a monoclonal antibody to an early nuclear antigen. Am J Clin Pathol 91 : 695-700, 1989.

Hepatic cytomegalovirus involvement in autopsy cases.

Hepatic involvement of cytomegalovirus (CMV) was studied in 15 autopsy cases with disseminated or solitary CMV infection. Formalin-fixed, paraffin-emb...
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