Case Reports

A Kaposi-like Variant of Splenic Angiosarcoma Lacking Association With Human Herpesvirus 8

Tetuo Mikami, MD; Makoto Saegusa, MD; Fumiyuki Akino, MD; Daisuke Machida, MT; Keiichi Iwabuchi, MD; Shotaro Hagiwara, MD; Isao Okayasu, MD

● We report the case of a Kaposi-like variant of splenic Abdominal ultrasonography and computed tomography revealed angiosarcoma in a 28-year-old woman. The tumor featured splenomegaly (Figure 1, a), and she was admitted to Kitasato University Hospital, Sagamihara, Japan. Laboratory examination a Kaposi sarcoma–like spindle cell proliferation with slit 12 formation and markedly dilated spongelike vascular chan- revealed slight anemia (red blood cell count, 3.58 ϫ 10 /L; he- moglobin, 107 g/L; hematocrit, 0.32), and because a malignant nels filled with erythrocytes. Thirteen months following the lymphoma was initially suspected, splenectomy was performed. initial splenectomy, metastatic lesions were found in the The resected spleen weighed 530 g and showed diffuse dark- patient’s liver and bone marrow. The proliferating cells reddish enlargement (Figure 1, b) without nodular lesions. His- were positive for factor VIII–associated antigen and CD34. tologically, the splenic normal structure was entirely replaced by The human herpesvirus 8 genome, which is regarded as a diffuse proliferation of spindle cells arranged in a fascicular pat- diagnostic feature of Kaposi sarcoma, was not detected by tern with erythrocytes containing slit formations (Figure 1, c). polymerase chain reaction analysis. Although the histologic Occasionally, markedly dilated spongelike spaces filled with findings were similar, this Kaposi-like variant of splenic an- erythrocytes were seen (Figure 1, d). Nuclear pleomorphism was giosarcoma must be considered distinct from Kaposi sar- minimal. Several calcium deposits were found within the re- coma. maining splenic trabeculae. The accessory spleen, measuring 1.5 cm in diameter, also exhibited the spindle cell proliferation. (Arch Pathol Lab Med. 2002;126:191–194) Although Kaposi sarcoma was suspected from the pathologic findings, serologic examination revealed no human immunode- plenic angiosarcomas are the most common malignant ficiency virus infection. After 13 months, follow-up computed to- mography and gallium scintigraphy revealed focal hepatic and S primary nonlymphoid tumors, although their occur- 1,2 bone marrow involvement without clinical symptoms except for rence is rare. Microscopic findings are highly variable, slight anemia. Liver and bone marrow biopsies were performed. both within tumors and between cases. While a spongi- In the liver, spindle cells showed focal nodular proliferation, es- form or honeycomb-like proliferation of endothelial cells pecially along Gleason sheaths. Occasionally, the spindle cells in- is the most common finding, Kaposi sarcoma–like prolif- vaded the sinusoids, replacing the normal endothelium (Figure eration is infrequent.3,4 Recently, human herpesvirus 8 2, a). In the bone marrow, the spindle cells were located in the (HHV-8) was detected in a case of acquired immunodefi- myxoid mesenchyma and were separated from each other (Figure ciency syndrome (AIDS)-associated Kaposi sarcoma,5 and 2, b). She was then treated with adriamycin. Although the met- now it is considered to be present in all Kaposi sarcomas, astatic lesions had not completely disappeared, the patient was regardless of any association with AIDS.6 Furthermore, still alive 21 months after the first operation. HHV-8 DNA detection using polymerase chain reaction PATHOLOGIC FINDINGS (PCR) or in situ hybridization is now considered to be a Immunohistochemistry critical test for the diagnosis of Kaposi sarcoma.6,7 We en- countered a case of splenic angiosarcoma showing Ka- Tissue samples from the spleen were fixed in buffered posi-like spindle cell proliferation. We report here the ex- formalin and embedded in paraffin. To investigate the tu- amination of HHV-8 DNA by PCR analysis and discuss mor cell character, immunohistochemical staining of tissue differences between splenic angiosarcomas and Kaposi sections was performed using the labeled streptavidin-bi- sarcomas. otin method (commercial kit, Dakopatts, Glostrup, Den- mark), with anticytokeratin (monoclonal, clone CAM 5.2, REPORT OF A CASE Becton Dickinson, San Jose, Calif), antivimentin (monoclo- A 28-year-old housewife without any particular past individual nal, clone v9, Dako), anti-factor VIII–associated antigen or familial medical history experienced persistent left flank pain. (polyclonal, Dako), anti-CD34 (monoclonal, clone QBEND 10, Immunotech, Marseille, France), anti-CD68 (monoclo- Accepted for publication July 12, 2001. nal, clone PG-M1, Dako), anti–␣-smooth muscle actin From the Department of Pathology (Drs Mikami, Saegusa, Akino, (monoclonal, clone 1A4, Dako), antilysozyme (polyclonal, Iwabuchi, and Okayasu), Kitasato University School of Medicine, Sa- Dako), and anti–Ki-67 (polyclonal, Dako) primary anti- gamihara, Japan; and the Departments of Pathology (Drs Mikami, Sae- bodies. To compare the cell proliferation activity of the gusa, Akino, Iwabuchi, and Okayasu, and Mr Machida) and Hematol- ogy (Dr Hagiwara), Kitasato University Hospital, Sagamihara, Japan. tumor with data for vascular tumors, 11 cases of cavernous Reprints: Tetuo Mikami, MD, Department of Pathology, Kitasato Uni- hemangioma and 15 cases of hemangiosarcoma of scalp versity School of Medicine, 1-15-1, Kitasato, Sagamihara-si, Kanagawa, were retrieved from the pathology files of Kitasato Uni- 228-8555, Japan (e-mail: [email protected]). versity Hospital, and immunohistochemistry for Ki-67 an- Arch Pathol Lab Med—Vol 126, February 2002 Splenic Angiosarcoma—Mikami et al 191 Figure 1. a, Splenomegaly observed by abdominal computed tomography. b, Cut section of the resected spleen. Note diffuse swelling with a dark reddish appearance. Each division of the scale is 5 mm. c, Spindle cells are arranged in a fascicular pattern with formation of slitlike channels, mimicking Kaposi sarcoma (hematoxylin-eosin, original magnification ϫ200). d, Markedly dilated spaces filled with erythrocytes and lined by endothelium (hematoxylin-eosin, original magnification ϫ40). tigen was performed. At least 1000 tumor cells were ex- ments were noted (Figure 4). Some cells contained intra- amined for each case, and the percentage of positively cytoplasmic lumina filled with erythrocytes. stained nuclei was calculated as the labeling index. Polymerase Chain Reaction Analysis for HHV-8 Proliferating spindle cells and lining cells of the cavern- ous spaces showed immunoreactivity for factor VIII–as- DNA was extracted from fresh frozen tumor tissue and sociated antigen and CD34 (Figure 3, a and b). They were sections of formalin-fixed, paraffin-embedded tumor tis- negative for cytokeratin, CD68, ␣-smooth muscle actin sues by proteinase K digestion, phenol-chloroform extrac- (Figure 3, c), and lysozyme. The Ki-67 labeling index for tion, and ethanol precipitation. Using the primer set (5Ј- the present case was 5.4%, while the labeling indices of AGCCGAAAGGATTCCACCAT-3Ј and 5Ј-TCCGTGTTGT 8 the cavernous hemangiomas and the hemangiosarcomas CTACGTCCAG-3Ј), 35 cycles of PCR amplification of a of the scalp were 1.3% Ϯ 1.5% (mean Ϯ standard devia- section of the HHV-8 genome were carried out. These tion) and 20.8% Ϯ 10.9%, respectively. primers amplify a 233-base pair (bp) fragment located from base pair 987 to base pair 1219 of the reported HHV- Electron Microscopy 8 sequence.5 As a substitute for template DNA, distilled water was added to the reaction solution as a negative Electron microscopy was performed with fresh tissues control. As a positive control, DNA samples from 3 cases fixed in glutaraldehyde and postfixed in osmium tetrox- of AIDS-associated Kaposi sarcoma were used. ide. Ultrathin sections were stained with uranyl acetate After electrophoresis, the agarose gels were stained and lead citrate and examined with a transmission elec- with ethidium bromide and visualized by ultraviolet light. tron microscope (H-600, Hitachi, Tokyo, Japan). While the PCR product of 233 bp was detected for the 3 The proliferating spindle cells were observed to form samples from Kaposi sarcomas, no product of this size primitive junctional complexes with neighboring cells. Pi- was seen with our present case (Figure 5). nocytotic vesicles were found beneath the plasma mem- branes, and cytoplasmic organelles were sparse, although COMMENT swollen mitochondria, rough endoplasmic reticulum, ly- Splenic angiosarcoma is a rare tumor, and most details sosomes, and occasional collections of intermediate fila- concerning this tumor have been published in case re- 192 Arch Pathol Lab Med—Vol 126, February 2002 Splenic Angiosarcoma—Mikami et al Figure 2. a, The histology of a liver biopsy specimen. Spindle cells are proliferating in the Gleason sheath (the arrowhead shows a branch of the hepatic artery) (hematoxylin-eosin, original magnification ϫ200). b, Histology of a bone biopsy. Spindle cells with atypical nuclei are sparsely located in the bone marrow cavity (hematoxylin-eosin, origi- nal magnification ϫ400). ports. In 1993, Falk et al3 reported a clinicopathologic study of 40 collected cases. More recently, Neuhauser et al4 described the clinicopathologic findings with a detailed immunohistochemical study of 28 cases. According to these 2 articles, the histology of splenic angiosarcoma is highly variable. Although the most common type features a spongiform proliferation of endothelial cells forming ir- regular capillary networks. Other pseudosinusoidal, pseu- dopapillary, and spindle cell sarcoma–like patterns have been documented.3,4 Neuhauser et al4 reported that only 2 of their collected cases showed Kaposi sarcoma–like spin- Figure 3. Immunohistochemical findings for the tumor. The spindle dle cell proliferations with slitlike spaces. In the present cells are immunopositive for (a) factor VIII–associated antigen and (b) case, the tumor was composed of 2 characteristic compo- CD34. On the other hand, the spindle cells are immunonegative for nents: spongelike, dilated, thin-walled endothelium and (c) ␣-smooth muscle actin (diaminobenzidine and hematoxylin, origi- Kaposi sarcoma–like spindle cells. Immunohistochemical- nal magnification ϫ400). ly, the spindle cells were immunopositive for factor VIII– associated antigen and CD34 and were immunonegative liferating cells. We therefore concluded that the present for ␣-smooth muscle actin, although Kaposi sarcomas gen- case is a rare variant of the splenic angiosarcoma, a Ka- erally express ␣-smooth muscle actin but lack expression posi-like variant. of factor VIII–associated antigen.9,10 Ultrastructural find- In the literature, although some controversial findings ings confirmed an endothelial differentiation of the pro- were reported,11 most studies were unable to detect HHV- Arch Pathol Lab Med—Vol 126, February 2002 Splenic Angiosarcoma—Mikami et al 193 endothelium. The authors came to a diagnosis of Kaposi sarcoma only from the former component. Their tumor metastasized to the liver, the mesenterium, the pleura, and the lymph nodes. Although the authors did not investigate for HHV-8 DNA (the report was published before the dis- covery of the virus), we believe that the splenic tumor they reported belongs to the same category as our case because of the similar histologic findings and the malignant be- havior. The symptoms in our patient were slight anemia and left flank pain. According to the collected series of Falk et al,3 the major symptoms are abdominal pain (83%) and cytopenia (91%), including anemia, thrombocytopenia, and pancytopenia. A rare but important presentation of splenic angiosarcoma is spontaneous splenic rupture (13%). The prognosis of splenic angiosarcoma is poor. Cal- culated from the cases in the reports of Falk et al3 and Neuhauser et al,4 1- and 3-year survival rates were 52.1% and 13.5%, respectively. The prognosis of the Kaposi-like variant remains to be determined. In earlier reports, nuclear atypia varied from case to Figure 4. Ultrastructural findings for spindle cells of the tumor. Pi- case. In the present lesion, nuclear atypia was minimal, nocytotic vesicles are seen beneath the plasma membrane (arrow). Col- especially in the spongelike component. However, the cell lections of intermediate filaments are also illustrated (arrowheads). n proliferation rate was between those for hemangiosarco- indicates nucleus; bar, 1000 nm (original magnification ϫ12 000). mas of scalp and cavernous hemangiomas. Pathologists should bear in mind that splenic angiosarcomas can show minimal nuclear atypia. In conclusion, we report here a rare variant of splenic angiosarcoma. Although histologically similar to Kaposi sarcoma in part, it appeared to be a distinct entity because HHV-8 DNA was not detected. We conclude that it should be treated as a Kaposi-like variant of splenic angiosarco- ma. We thank M. Koike, MD, and S. Moriyama, MD, for providing DNA from Kaposi sarcomas in patients with acquired immuno- deficiency syndrome. We also thank the staff of the Electron Mi- croscope Centre, Kitasato University School of Medicine (Saga- mihara, Japan), and M. Yokozawa for their expert technical sup- port. References 1. Rosai J. Ackerman’s Surgical Pathology. 8th ed. St Louis, Mo: Mosby-Year Figure 5. Polymerase chain reaction (PCR) analysis of DNA extracted Book Inc; 1996:1775–1796. from the tumor. Lanes 1 and 2, DNA extracted from paraffin-embedded 2. Smith VC, Eisenberg BL, McDonald EC. Primary splenic angiosarcoma: case tissue; lanes 3 and 4, DNA from frozen tissue; lanes 5 through 7, DNA report and literature review. Cancer. 1985;55:1625–1627. from Kaposi sarcomas; lanes 8 and 9, DNA from malignant lymphomas 3. Falk S, Krishnan J, Meis JM. Primary angiosarcoma of the spleen: a clini- as negative controls; and lane 10, distilled water instead of template copathologic study of 40 cases. Am J Surg Pathol. 1993;17:959–970. 4. Neuhauser TS, Derringer GA, Thompson LDR, et al. Splenic angiosarcoma: DNA. In PCR for human herpesvirus 8, positive bands (233 bp) are a clinicopathologic and immunophenotypic study of 28 cases. Mod Pathol. 2000; evident in lanes 5 through 7 in the upper panel. The lower panel shows 13:978–987. the PCR for ␤-globin (product size, 250 bp) as positive controls. 5. Chang Y, Cesarman E, Pessin MS, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science. 1994;266:1865– 1869. 6. Cathomas G. Human herpes virus 8: a new virus discloses its face. Virchows 8 DNA in mesenchymal and vascular tumors other than Arch. 2000;436:195–206. Kaposi sarcoma, including hemangiomas, hemangioen- 7. Brooks LA, Wilson AJ, Crook T. Kaposi’s sarcoma-associated herpesvirus dotheliomas, infantile capillary hemangioma, and heman- (KSHV)/human herpesvirus 8 (HHV8): a new human tumour virus. J Pathol. 1997; 182:262–265. 6 giosarcoma. Identification of HHV-8 DNA is now regard- 8. Alkan S, Eltoum IA, Tabbara S, Day E, Karcher DS. Usefulness of molecular ed as the diagnostic criterion for Kaposi sarcoma.6 In the detection of human herpesvirus-8 in the diagnosis of Kaposi sarcoma by fine- present case, HHV-8 DNA was not detected with PCR needle aspiration. Am J Clin Pathol. 1999;111:91–96. 9. Weich HA, Salahuddin SZ, Gill P, Nakamura S, Gallo RC, Folkmann J. AIDS- analysis. Accordingly, a distinct entity of splenic angio- associated Kaposi’s sarcoma-derived cells in long-term culture express and syn- sarcoma—different from Kaposi sarcoma but with similar thesize smooth muscle alpha-actin. Am J Pathol. 1991;139:1251–1258. histologic findings—may exist. 10. Enzinger FM, Weiss SW. Soft Tissue Tumors. 3rd ed. St Louis, Mo: Mosby- 12 Year Book Inc; 1995:658–669. To our knowledge, Sarode et al reported the only case 11. McDonagh DP, Liu J, Gaffey MJ, Layfield LJ, Azumi N, Traweek ST. De- of splenic Kaposi sarcoma to date. The histologic compo- tection of Kaposi’s sarcoma-associated herpesvirus-like DNA sequences in angio- nents of the case, judging from the description and the sarcoma. Am J Pathol. 1996;149:1363–1368. 12. Sarode VR, Datta BN, Savitri K, Singh K, Bhasin D. Kaposi’s sarcoma of photographs, comprised spindle cell proliferation and spleen with unusual clinical and histologic features. Arch Pathol Lab Med. 1991; thin-walled, dilated vascular channels lined by flattened 115:1042–1044.

194 Arch Pathol Lab Med—Vol 126, February 2002 Splenic Angiosarcoma—Mikami et al