ctinicat and Experimentat Dermatotogy 1992; 17: 145-151.
The St John's Oration 1990. Klaus Wolff delivered tlie St John^s Oration for 1990, 'The enigma of Kaposi's sarcoma: an answer at tasl'.
145
146
K.WOLFF
The St John's Oration, 1990
The enigma of Kaposi's sarcoma: an answer at last? K.WOLFF Department of Dermatology I, University of Vienna, Alser Strasse 4, A-1090 Vienna, Austria Accepted far publication 1 September 1991
Kaposi's sarcoma (KS)' was of little interest to the general medical community until its endemic occurrence in Africa'-^ and in immunosuppressed organ transplant recipients*-^ became known and its epidemic spread among young, male homosexuals''' and thus its association with what later became known as AIDS attracted world-wide attention. KS is now considered to be one of the most interesting angioproliferative processes in medicine. Current classification'*'' based on epidemiological and clinical criteria distinguishes four different forms of KS to which a fifth'" can be added (Table 1); pathogenetic considerations include a genetic predisposition, hormonal, neural and angiogenic factors, disturbances of the reticuloendothelial and immune systems, systemic carcinogens and infectious agents.*' A discussion of our current understanding of KS should thus focus on the following questions:
(a) Are all forms of Kaposi's sarcoma one and the same disease? (b) Is Kaposi's sarcoma a malignant neoplasm? (c) What is the nature ofthe Kaposi's cell? (d) What induces proliferation of these cells? (e) Are there leads to the aetiological agents that induce this proliferation?
Is Kaposi's sarcoma a uniform entity? Clinical (Table 2), histopathological and demographic (Table 3) data suggest that all five forms of KS represent ditferent phenotypes within a uniform disease spectrum. Only the association of certain types of KS with immunosuppression does not fit clearly into this scheme but it correlates with the degree of progression and a poor prognosis. Is KS a malignant tumour?
Table 1. Classitication of KS
The rapidly progressive forms of KS, in particular KS associated with AIDS and iatrogenic immunosuppression or the juvenile form ofthe endemic African variant, suggest a malignant process as does the histopathology of the advanced tumour stage.^ On the other hand, the early macular and plaque lesions do not exhibit features of malignancy and the faet that KS lesions are composed of
(ilassieal KS Kndemic .African KS KS in iairogenic immunosuppression AinS-associatcd KS Endemic Mediterranean KS
Table 2. Kaposi's sarcoma: clinical features
Type Oassical K S Endemic African K S KS in immunosuppression AIDS-KS Endemic ,\tcditcrranean KS
Skin
Lymphnodes
(lastrtiintestinal iract
(ither internal organs
C bourse
slow slow/rapid slow/rapid regressive rapid slow
THE ENIGMA OF KAPOSI'S SARCOMA
147
Table 3. Kaposi's sarcoma: demographic characteristics Determinatits Type Classical KS Endemic African KS KS in immunosuppression AIDS-KS Endemic Mediterranean KS
Age
Sex
Old age All age groups Adults Adults Higher age group
m> > >f m> > >f
Geographic
Ethnic
Behaviour
4-4-
+
m>f
m(f) m>f
multiple, different cell types (spindle cells, endothelial cells, fibroblasts, dermal factor Xlll-positive dendrocytes, T-lymphocytes) fails to support the concept of a monoclonal neoplastic process. Further features arguing against malignancy are the absence of aneuploidy, the multicentric origin of KS, its course and the predictability of its progression and the involvement of skin and internal organs as well as the pattern of lymph node and mucosal infiltration." Finally, spontaneous remissions in immunosuppressed patients upon termination of immunosuppression and the fact that true metastasis has never been unequivocally proven for this condition make it difficult to accept KS as a truly neoplastic process. A number of authors,"'^ including ourselves, therefore do not consider KS to be a true cancer but a potentially reversible hyperplasia.
pluripotential mesenchymal cells, fibroblasts, endothelial cells'* or FXIII+ dermal dendrocytes.'^ Immunophenotyping of KS lesions has revealed the striking resemblance of spindle cells to the endothelial cells of lymphatics'^"'^ and our own investigations have confirmed and expanded on this notion'" (Table 4). Electron microscopic investigations have emphasized the absence of Weibel-Palade bodies from KS cells, a discontinuous basal lamina and an absence of pericytes '^ and a transition from spindle cells partially surrounded by endothelial basal lamina to slits lined by typical lymphatic endothelium. ^'^ We therefore consider that KS cells are derived from the endothelium of lymphatic vessels^" but it should be pointed out that other authors have detected immunophenotypic markers of blood vessel endothelium on spindle cells.'**
What is the nature ofthe cells proliferating in KS?
What drives the proliferation of Kaposi's sarcoma cells?
There is no question that KS lesions are, at least partially, angioproliferative in nature, On the other hand there is no agreement as to the origin of the spindle cells that dominate the histopathological appearance of KS lesions. These have been variably interpreted as arising from
This question can best be analysed by studying growth requirements and growth kinetics of KS cells in vitro and most investigations have therefore focused on factors that are known to stimulate the growth of endothelial cells. A
Tabte 4. Immunophenotype of KS celt* Normal human skin MoAb PAL-E EN-4 Anti-von Willehrand faetor OKM-5 (anti-CD36) Anti-laminin Anti-collagen type-4 Anti-desmin Anti-vimentin
Endothelium of mature: Blood vessels Lymphatic vessels
4- + + -f
4-4-
KS lesions Endothelium of clefts/slits
Spindle cells
-1-
4-
44-
4- + 4- + _ 4-4-
44-4-
4-4-
4-4-
—: negative; (4-): occasionally weakly positive; 4-: weakly positive; 4- -f-: strongly positive. * Rappersberger et al.^^
148
K.WOLFF
major breakthrough was provided by Nakamura et al. in 1988'"* who observed that retrovirus-infected CD4^ ceils produce growth factors which permit the long-term growth of spindle-like cells from pleural effusions and pulmonary KS lesions of patients with AIDS. Salahuddin et al.'^" have shown that conditioned media obtained from HLTV-II-infected CD4+ cells support the growth of (AIDS-) KS cells which in turn produce factors that stimulate both their own growth in an autocrine and that of other endothelial cells in a paracrine fashion. Such cultured cells liberate angiogenic factors which, upon transplantation of these cells onto the chorioallantoic membrane, produce angiogenic lesions, and after transplantation into nude mice, angiomatous tumours.^"^ Of the various cytokines and growth factors tested, bFGF,^' IL-1,^' IL-6^^ and tat protein" have emerged as important candidates responsible for the angiogenic activity of these pulmonary-tissue derived (AIDS)-KS lines but we have not been very successful in propagating cells derived from cutaneous KS lesions of HIV-infected and HIV-negative patients with these factors in vitro. On the other hand, conditioned medium derived from an EBV-transformed dendritic B-cell line,^"* strongly stimulated the growth of cells derived from cutaneous KS lesions and we have been able to show that the factor(s) responsible for this propagation was (were) inhibited by heparin-sepharose but not by anti IL-la, anti-IL-6 or anti-bFGF monoclonal antibodies.^'' The nature of this factor, which has a molecular weight of about 52 kD, has so far not been clarified but an analysis of the immunophenotypic marker profile ofthe cultured cells has shown an excellent correlation with the immunophenotype of KS cells in vivo., indicating that the propagated cell lines indeed represent KS cells. What about the factors that have been shown by others^''^^ to exert growth stimulatory activity in vitro under conditions chosen by these investigators? SchultzeOsthofft'/ al. have failed to find bFGF mRNA-expression on KS spindle cells in situ}" Our own RNA-RNA in-situ hybridization studies have taught us that whereas bFGF and KFGF-mRNA are indeed expressed in some cells of cutaneous KS lesions, these lesions also contain a considerable amount of IL-6 mRNA message.^'' This, however, is localized predominantly in the tumour periphery and in the interstitial tissue adjacent to and within KS lesions. It correlates with the rate of progression of such lesions in that it is predominantly found in rapidly growing tumours. We have recently shown tbat IL-6 mRNA can be localized to activated dermal dendrocytes^'' and the absence of ICAM-1 and ELAM-1/ ENA-1 expression by KS cells in situ has suggested to us that IL-1 or TNFct//? do not significantly contribute to the growth of these lesions.^'' As KS lesions express a high level of monocyte chemo-attractant protein 1 (MCP-1)^' which is also found within the basal-cell layer of
tbe epidermis, we presently assume that the growth of KS lesions is a self-amplifying process driven by reciprocal stimulation of KS cells and dermal dendrocytes attracted to the lesions by MCP-1.^' Returning to the tat-protein which has been shown by Ensoli et alP to represent an important growth factor for certain KS cell lines, there are a number of experiments and observations that permit seme fascinating speculation. Vogel et al?^ were able to show that HlV-tattransgenic mice develop tumours which clinically and histologically are similar to KS lesions in man. It is interesting that transgenic mice of both sexes express tatmRNA in the skin but KS-like lesions occur only in male animals which is analogous to the situation of AIDSassociated KS. In addition, it was shown, that tat-mRNA is not expressed in cell lines derived from these tumours. Expanding on these experiments Ensoli et alP provided evidence that tat protein is released by HIV-1 infected H9-cells and tat-transfected COS-1 cells and that not only supernatants containing tat-protein, but also recombinant tat-protein, support the growth of AIDS-KS cells in vitro. Furthermore, this propagation could be inhibited by anti-tat-antibodies. These observations appear to suggest that a viral regulatory gene product is released as a biologically active protein which can serve as a stimulator of cell growth in its own right.^-^ If we now interpret these findings to fit the scenario in HIV-infected patients it will be remembered that AIDSKS lesions contain CD4+ cells^** that are potentially HIVinfected and therefore could release tat-protein which in turn could stimulate the growth of KS lesions. We also know that in HIV-positive patients replication of HIV occurs in epidermal Langerhans cells^" which therefore also could represent a source for tat protein-mediated growth stimulation of KS. As KS lesions arise in the subepidermal dermis it does not appear unreasonable to speculate that signals for KS proliferation could be derived from HIV-infected epidermal Langerhans cells. What is the aetiological agent of KS? A viral aetiology of KS has been discussed for years and the potential role ofthe tat-gene in the growth promotion of this tumour focuses new interest on a possible viral aetiology. In the past, CMV has been discussed as a potential aetiological factor but evidence to support this contention has not been forthcoming.^'^' In the early years of the HIV epidemic KS was also aetiologically linked to HIV, particularly in view ofthe fact that the risk of developing Kaposi's sarcoma is approximately 20000 times greater in AIDS patients than in the general population and approximately 300 times greater than in other types of immunosuppression.^- On the other hand, HIV genes or gene products havenot been detected in KS lesions and there is no correlation hetween the appearance
THE ENIGMA OF KAPOSI'S SARCOMA and course of KS and the degree of immunosuppression.** Further features that argue against a causal relationship between HIV and KS are the observation that more than 90% of KS in HIV-infected individuals occur in male homosexuals and that most HIV-infected individuals do not develop KS; KS also occurs in HIV-negative male homosexuals and women who only rarely have KS, develop this tumour when their HIV-positive partners are bisexual or i.v. drug users. It is also quite striking that the incidence of KS in HIV-positive homosexuals has fallen from 42% in 1983 to 13% in 1988 despite the fact that the incidence of HIV infection has increased.'^ These and other additional reasons have suggested to Beral et al.^^ that KS in AIDS patients is possibly the result of the infection with another sexually transmissible agent that may be concomitantly transmitted with HIV. Indeed, electron microscope investigations have shown that KS cells in AIDS-associated KS^-* ^'' contain intracellular
149
inclusions—tubuloreticular structures (TRS) and cylindrical-confronting cisternae (CCC)—which today are interpreted as cellular reaction products to viral infections.^* TRS and CCC are also abundantly present in KS cells of the endemic Mediterranean KS variant'" and classical KS. They can be induced in vitro by interferon (x/^^*'"-'^and this raises the question whether KS could be due to a hitherto unknown retroviral agent which is different from HIV. We have recently found, by electronmicroscopy, virus particles in KS lesions'" that morphologically unequivocally correspond to oncovirinae.^^ These particles occur both as immature and mature viral structures intra- and extracellularly in KS lesions (Fig. 1) and there is morphological evidence suggesting an uptake of such particles via coated pits and their segregation through a process of budding into intracellular vacuoles (Fig. 1). Although so far we have no molecular biological and biochemical evidence to prove the presence of
Figure 1. Retrovirat particles identitied in KS lesions, (a) immature virus with 7-10 mm spikes al the enveloping membrane (arrows) (x 120 000); (h) extracellular virus particte (arrow) close to a KS cell (x 60 000); (c) uptake of retrovirus (arrow) hy coated pit {bold arrow) ( X 60 000); (d) particte (arrow) within a cytoptasmic vacuote of a KS cetl ( x 66 000). (Reproduced from Rappersberger el al.'^\ Journal ofInvestigative Dermatology., with kind permission, Elsevier Science Pubtishing Co. Inc NewYork,)
150
K.WOLFF
retroviruses in KS lesions, the morphological data leave no doubt that retroviral particles do occur on KS. These particles have so far been identified reproducibly in endemic Mediterranean"* and classical KS (unpublished results) so that the retroviruses originally found in HIVassociated KS—originally interpreted to represent HIV*^'*^—could now possibly be interpreted in a different light. Proof of the pathogenetic relevance of these viruses would support the hypothesis of Dictor and Jarplid''-^ which considers KS to be a human retroviral analogue of fowl haemangiomatosis which is caused by a retrovirus ofthe lymphoid leukosis group. Outlook The concept of Kaposi's sarcoma as a retroviral process is indeed fascinating. However, I wish to emphasize that evidence available to date is entirely based on morphological data and merely indicates that retroviral particles are present in KS lesions. We have so far been unsuccessful in characterizing this retrovirus by molecular and biochemical methods and the decisive question therefore remains whether these particles have any pathogenetic significance. Proof of this would however permit us to paint a scenario in which such viruses are transmitted either sexually or non-sexually and in the case of AIDS together with, or independently of, HIV and to initiate cellular processes that directly lead to a transformation and the proliferation of endothelial cells. Indeed it has been shown by Delli-Bovi and Basilico'*'' and by Lo and Liotta^^ that KS-DNA transforms NIH/3T3 cells and that such transformants produce angiosarcomatous lesions in nude mice. Another role of KS retroviruses could consist of the stimulation of the production and secretion of angiogenic and growth factors by endothelial cells and dermal dendritic cells which in turn would initiate a self perpetuating cycle of cellular proliferation by paracrine or autocrine secretion. These lines of thought are presently entirely speculative in nature but they do provide an interesting model for further research.
Acknowledgments I would like to thank the following who have, through their research, made important contributions to this oration: Professor Georg Stingl, Dr Klemens Rappersberger, Dr Reinhard Gillitzer, Dr Rudolf Berger, Dr Erwin Tschachler, Dr Eva Zonzits, Dr Angelos Hatzakis, Dr Dean L.Mann, Dr Robert J.Biggar. References t, Kaposi M. Idiopathisches rnultiptes pigmentsarkom der haut. Archives of Dermatology and Syphilis (Prag) t872; 4: 265-273. 2. Stavin G, Cameron HMcD, Singh H, Kaposi's sarcoma in
mainland Tanzania: a report of 117 cases, British Journal of Camer 1969; 23: 349-357, 3. Taylor JF, Templeton AC, Vogel CL, Ziegter JL, Kyalwazi SK. Kaposi's sarcoma in Uganda: a clinico-pathological study. International Journal oj'Cancer 1971; 8: 122-135. 4. Penn I. Kaposi's sarcoma in organ transplant recipients: report of 20 cases. Transplantation 1979; 27: 8-11. 5. Shumeli D, Shapira Z, Yussim A, Nakache R, Ram Z, Shaharabani E. The incidence of Kaposi sarcoma in renal transplant patients and its relation to immunosuppression. Transplant Proceedings 1989; 21: 3209-3210. 6. Hymes KB, Cheung T, Greene JB, Prose NS, Marcus A, Ballard H, William DC, Laubenstein LJ. Kaposi's sarcoma in homosexual men—report of eight cases. Eancet 1981; ii: 598-600. 7. Friedman-Kien AE, Lauhenstein LJ, Ruhinstein P, BuimoviciKlein E, Marmor M, Stahl R, Spigland I, Kim KS, Zolla-Pazner S. Disseminated Kaposi's sarcoma in homosexual men. Annals of Internal Medicine 1982; 96: 693-700. 8. Safai B. Kaposi's sarcoma: an overview of classical and epidemic (brms. In: Broder S etl, AIDS. Modern Concepts and Therapeuti Challenges. Marcel Dekker, Inc.: New York and Basel, 1987: 205218, 9. Ziegler JL, Dorfman RF (eds), Kaposi's Sarcoma. Pathophysiology and Clinical Management. Marcel Dekker, Inc.: New York and Basel, 1988, 10. Rappersberger K, Tschachler E, Zonzits E, Gillitzer R, Hatzakis A, Kaloterakis A, Mann DL, Popow-Kraupp T, Biggar J, Berger R, Stratigos J, Wolff K, Stingl G. Endemic Kaposi's sarcoma in human immunodeficiency virus type 1-sero-negative persons: demonstration of retrovirus-like particles in cutaneous lesions. Journal of Investigative Dermatology 1990; 95: 371-381. 11. Costa J, Rabson AS. Generalized Kaposi's sarcoma is not a neoplasm, Eancet 1983; i: 58. 12. Brooks JJ. Kaposi's sarcoma: a reversible hyperplasia. Lancet 1986; ii: 1309-1311. 13. NickolofF BJ, Griffiths CEM. Factor Xllla-cxpressing dermal dendrocytes in AIDS-associated cutaneous Kaposi's sarcomas. Science 1989; 243: 1736-1737. 14. Scully PA, Steinman HK, Kennedy C, Truehlood K, Frisman DM, Voland JR. AIDS-related Kaposi's sarcoma displays differential expression of endothelial surface antigens. American Journal of Pathology 1988; 130: 244 251. 15. Green TL, Meyer JR, Daniels TE, Greenspan JS, de Souza Y, Kratner RH, Kaposi's sarcoma in AIDS: hasement membrane and endothelial cell markers in late stage lesions. Journal of Oral Pathology 1988; 17: 266 272. 16. Nadimi H, Saatee S, Armin A, Toto PD. Expression of endothelial cell markers PAL-E and EN-4 and Ia antigens in K.zpQ%i'^ ^'urcomi. Journal of Oral Pathotogy 1988; 17:416-420 17. McNutt NS, Fletcher V, Conant MA. Early lesions of Kaposi's sarcoma in homosexual men. An ultrastructural comparison with other vascular proliferations in skin. American Journal of Pathology 1983; 111:62-77. 18. KrafFert CA, Planus L, Penneys NS. Kaposi's sarcoma: further immunohistologic evidence of a vascular endothelial origin. Journal of Investigative Dermatotogy 1991; 96: 598, 19. Nakamura S, Salahuddin SZ, Biherfeld P, Ensoli B, xMarkham PD, Wong-Staal F, Gallo RC, Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4"'^ cells. Science 1988; 242: 426-430. 20. Salahuddin SZ, Nakamura S, Biberfeld P, Kaplan MH, Markham PD, Larsson L, Gallo RC. Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro. Science 1988; 242: 430-433. 21. Ensoli B, Nakamura S, Salahuddin SZ, Biberfeld P, Larsson L, Beaver B, Wong-Staal F, Gallo RC. AIDS-Kaposi's sarcoma-
THE ENIGMA OF KAPOSI'S SARCOMA
22.
23.
24. 25.
26.
27.
28. 29.
30.
31. 32.
derived cells express cytokines with autocrine and paracrine growth effects. Science 1989; 243: 223-226, Miles SA, Rczai AR, Salazar-Gonzalez JF, Van der Meyden M, Stevens RH, Logan DxVl, Mitsuyasu RT, Taga T, Hirano T, Kishimoto T, Martinez-Maza O. AIDS Kaposi sarcoma cells produce and respond to interleukin 6, Proceedings ofthe Nationat Academy of Sciences ILSA 1990; 87: 4068-4072, Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F, Tat-protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature 1990; 345: 84-86, Berger R, Purtsche M, Tong D, WolfTK, Stingl G. A tibrohlast growth factor-like activity secreted by a dendritic B cell line. Archives of Dermatotogy Research 1991; 283: 63. Schulze-Osthoff K, Goerdt S, Sorg C, Expression of hasic fibrnhlast growth factor (bFGF) in Kaposi's sarcoma: an immunohistologic study. Journal oj'Investigative Dermatological 1990; 95: 238-240. Gillitzer R, Miiller C, Wolff K, Stingl G, Berger R. High in situ expression of IL-6 mRNA in Kaposi's sarcoma: a possihie role in
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33. Gyorkey F, Sinkovics JG, Gyorkey P, Tubulo-reticular structures in Kaposi's sarcoma. Lancel 1982; ii: 984 985. 34. Sidhu GS, Stahl RE, El-Sadr W, Zolla-Pazner S. Ultrastructural markers of AIDS, Lancet 198.3; i: 990-991. 35. Sidhu GS, Stahl RE, El-Sadr W, Cassai ND, Forrester ExM, Zoila-Pazner S. The acquired immunodeficiency syndrome: an ultrastructural study. Human Pathology 1985; 16: 377-386. 36. I.uu J, Bockus D, Remington F, Bean MA, liammar SP. Tuhuloreticular structures and cylindrical confronting cisrernae: a review. Human Pathology 1989; 20: 617-627. 37. Yoffe B, Petrie BL, Noonan CA, Hollinger FB. In vivo and in vitro ultrastructural alterations induced by human immunodeficiency virus in human lymphoid cells. Eahoratory Investigations 38. Orenstein JM, Preble OT, Kind P, Schulof R. The relationship of serum alpha-interferon and ultrastructural markers in HIVseropositive individuals. Ultrastructural Pathology 1987; 11: 673-679. 39. Gelderhlom HR, Ozel M, Pauli G. Morphogenesis and morphology of HIV structure-function relations. Archives of Vtrotogy 1989; 106: 1-13. oncogenesis. Archives of Dermatological Research 1991; 283: 40. Gyorkey F, Sinkovics JG, Melnick JL, Gyorkey P. Retroviruses 57-58. in Kaposi-sarcoma cells in AIDS. ?^ew Engtand Journal of Gillitzer R, Stingl G, Burg G, Wolff K, Berger R. High levels of Medicine 1984; 311: 1183-1184. mRNA expression of monocyte chemoattractant protein-1 41. Schenk P, Konrad K. Ultrastructure of Kaposi's sarcoma in (MCP-l) and interleukin 6 (IL-6) in Kaposi's (KS) sarcoma acquired immune deficiency syndrome (AIDS). Archives of lesions in situ. Journal of Investigative Dermatotogy 1991; 96: 598, Otorhinotaryngology 1985; 242: 305-313. Vogel J, Hinrichs SH, Reynolds'RK, Luciw PA, Jay G, The HIV 42. Schenk P. Retroviruses in Kaposi's sarcoma in acquired immune tat gene induces dermal lesions resembling Kaposi's sarcoma in deficiency syndrome (AIDS). Acta Otolaryngology 1986; 10!: transgenic mice. Nature 1988; 335: 606-611, 295-298." Gillitzer R, Berger R, Rappersberger K, Wolff' K, Stingl G, 43. Dictor M, Jarplid B, The cause of Kaposi's sarcoma: an avian Characterization of lymphocyte infiltrates in endemic vs epidemic retroviral -innXo^. Journat ofthe American Academy ofDermatology Kaposi's s-ivcom-i. Journat of Investigative Dermatotogy 1989; 92: 1988; 18: 398-402. 434. 44. Delli-Bovi P, Basitico C. Isolation of a rearranged human Tschachler E, Groh V, Popovic M, Mann DL, Konrad K, Safai transforming gene following transfection of Kaposi sarcoma B, Ezon L, di Marzo-Veronese F, Woltl'K, Stingl G. Epidermal DNA. Proceedings of lhe National Academy of Science USA 1987; Langerhans cells—a target for IITLV-III/LAV infection. Jour84: 5660-5664. nat of Investigative Dermatology 1987; 88: 233-237, 45. Lo SC, Liotta LA. Vascular tumors produced by NIH/3T3 cells Rudlingcr R, Neumann-Haefelin D. Zytomcgalieviren und transfected with human AIDS Kaposi's sarcoma DNA. American Kaposi-Sarkom, Hautarzt 1987; 38: 573-576. Journal of Pathology 1985; 118: 7-13. Beral V, Peterman TA, Berkelman RL, Jaffe HW, Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet 1990; 335: 123-128.
The St John's Oration 1990. Klaus Wolff delivered tlie St John^s Oration for 1990, 'The enigma of Kaposi's sarcoma: an answer at tasl'.
145
146
K.WOLFF
The St John's Oration, 1990
The enigma of Kaposi's sarcoma: an answer at last? K.WOLFF Department of Dermatology I, University of Vienna, Alser Strasse 4, A-1090 Vienna, Austria Accepted far publication 1 September 1991
Kaposi's sarcoma (KS)' was of little interest to the general medical community until its endemic occurrence in Africa'-^ and in immunosuppressed organ transplant recipients*-^ became known and its epidemic spread among young, male homosexuals''' and thus its association with what later became known as AIDS attracted world-wide attention. KS is now considered to be one of the most interesting angioproliferative processes in medicine. Current classification'*'' based on epidemiological and clinical criteria distinguishes four different forms of KS to which a fifth'" can be added (Table 1); pathogenetic considerations include a genetic predisposition, hormonal, neural and angiogenic factors, disturbances of the reticuloendothelial and immune systems, systemic carcinogens and infectious agents.*' A discussion of our current understanding of KS should thus focus on the following questions:
(a) Are all forms of Kaposi's sarcoma one and the same disease? (b) Is Kaposi's sarcoma a malignant neoplasm? (c) What is the nature ofthe Kaposi's cell? (d) What induces proliferation of these cells? (e) Are there leads to the aetiological agents that induce this proliferation?
Is Kaposi's sarcoma a uniform entity? Clinical (Table 2), histopathological and demographic (Table 3) data suggest that all five forms of KS represent ditferent phenotypes within a uniform disease spectrum. Only the association of certain types of KS with immunosuppression does not fit clearly into this scheme but it correlates with the degree of progression and a poor prognosis. Is KS a malignant tumour?
Table 1. Classitication of KS
The rapidly progressive forms of KS, in particular KS associated with AIDS and iatrogenic immunosuppression or the juvenile form ofthe endemic African variant, suggest a malignant process as does the histopathology of the advanced tumour stage.^ On the other hand, the early macular and plaque lesions do not exhibit features of malignancy and the faet that KS lesions are composed of
(ilassieal KS Kndemic .African KS KS in iairogenic immunosuppression AinS-associatcd KS Endemic Mediterranean KS
Table 2. Kaposi's sarcoma: clinical features
Type Oassical K S Endemic African K S KS in immunosuppression AIDS-KS Endemic ,\tcditcrranean KS
Skin
Lymphnodes
(lastrtiintestinal iract
(ither internal organs
C bourse
slow slow/rapid slow/rapid regressive rapid slow
THE ENIGMA OF KAPOSI'S SARCOMA
147
Table 3. Kaposi's sarcoma: demographic characteristics Determinatits Type Classical KS Endemic African KS KS in immunosuppression AIDS-KS Endemic Mediterranean KS
Age
Sex
Old age All age groups Adults Adults Higher age group
m> > >f m> > >f
Geographic
Ethnic
Behaviour
4-4-
+
m>f
m(f) m>f
multiple, different cell types (spindle cells, endothelial cells, fibroblasts, dermal factor Xlll-positive dendrocytes, T-lymphocytes) fails to support the concept of a monoclonal neoplastic process. Further features arguing against malignancy are the absence of aneuploidy, the multicentric origin of KS, its course and the predictability of its progression and the involvement of skin and internal organs as well as the pattern of lymph node and mucosal infiltration." Finally, spontaneous remissions in immunosuppressed patients upon termination of immunosuppression and the fact that true metastasis has never been unequivocally proven for this condition make it difficult to accept KS as a truly neoplastic process. A number of authors,"'^ including ourselves, therefore do not consider KS to be a true cancer but a potentially reversible hyperplasia.
pluripotential mesenchymal cells, fibroblasts, endothelial cells'* or FXIII+ dermal dendrocytes.'^ Immunophenotyping of KS lesions has revealed the striking resemblance of spindle cells to the endothelial cells of lymphatics'^"'^ and our own investigations have confirmed and expanded on this notion'" (Table 4). Electron microscopic investigations have emphasized the absence of Weibel-Palade bodies from KS cells, a discontinuous basal lamina and an absence of pericytes '^ and a transition from spindle cells partially surrounded by endothelial basal lamina to slits lined by typical lymphatic endothelium. ^'^ We therefore consider that KS cells are derived from the endothelium of lymphatic vessels^" but it should be pointed out that other authors have detected immunophenotypic markers of blood vessel endothelium on spindle cells.'**
What is the nature ofthe cells proliferating in KS?
What drives the proliferation of Kaposi's sarcoma cells?
There is no question that KS lesions are, at least partially, angioproliferative in nature, On the other hand there is no agreement as to the origin of the spindle cells that dominate the histopathological appearance of KS lesions. These have been variably interpreted as arising from
This question can best be analysed by studying growth requirements and growth kinetics of KS cells in vitro and most investigations have therefore focused on factors that are known to stimulate the growth of endothelial cells. A
Tabte 4. Immunophenotype of KS celt* Normal human skin MoAb PAL-E EN-4 Anti-von Willehrand faetor OKM-5 (anti-CD36) Anti-laminin Anti-collagen type-4 Anti-desmin Anti-vimentin
Endothelium of mature: Blood vessels Lymphatic vessels
4- + + -f
4-4-
KS lesions Endothelium of clefts/slits
Spindle cells
-1-
4-
44-
4- + 4- + _ 4-4-
44-4-
4-4-
4-4-
—: negative; (4-): occasionally weakly positive; 4-: weakly positive; 4- -f-: strongly positive. * Rappersberger et al.^^
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major breakthrough was provided by Nakamura et al. in 1988'"* who observed that retrovirus-infected CD4^ ceils produce growth factors which permit the long-term growth of spindle-like cells from pleural effusions and pulmonary KS lesions of patients with AIDS. Salahuddin et al.'^" have shown that conditioned media obtained from HLTV-II-infected CD4+ cells support the growth of (AIDS-) KS cells which in turn produce factors that stimulate both their own growth in an autocrine and that of other endothelial cells in a paracrine fashion. Such cultured cells liberate angiogenic factors which, upon transplantation of these cells onto the chorioallantoic membrane, produce angiogenic lesions, and after transplantation into nude mice, angiomatous tumours.^"^ Of the various cytokines and growth factors tested, bFGF,^' IL-1,^' IL-6^^ and tat protein" have emerged as important candidates responsible for the angiogenic activity of these pulmonary-tissue derived (AIDS)-KS lines but we have not been very successful in propagating cells derived from cutaneous KS lesions of HIV-infected and HIV-negative patients with these factors in vitro. On the other hand, conditioned medium derived from an EBV-transformed dendritic B-cell line,^"* strongly stimulated the growth of cells derived from cutaneous KS lesions and we have been able to show that the factor(s) responsible for this propagation was (were) inhibited by heparin-sepharose but not by anti IL-la, anti-IL-6 or anti-bFGF monoclonal antibodies.^'' The nature of this factor, which has a molecular weight of about 52 kD, has so far not been clarified but an analysis of the immunophenotypic marker profile ofthe cultured cells has shown an excellent correlation with the immunophenotype of KS cells in vivo., indicating that the propagated cell lines indeed represent KS cells. What about the factors that have been shown by others^''^^ to exert growth stimulatory activity in vitro under conditions chosen by these investigators? SchultzeOsthofft'/ al. have failed to find bFGF mRNA-expression on KS spindle cells in situ}" Our own RNA-RNA in-situ hybridization studies have taught us that whereas bFGF and KFGF-mRNA are indeed expressed in some cells of cutaneous KS lesions, these lesions also contain a considerable amount of IL-6 mRNA message.^'' This, however, is localized predominantly in the tumour periphery and in the interstitial tissue adjacent to and within KS lesions. It correlates with the rate of progression of such lesions in that it is predominantly found in rapidly growing tumours. We have recently shown tbat IL-6 mRNA can be localized to activated dermal dendrocytes^'' and the absence of ICAM-1 and ELAM-1/ ENA-1 expression by KS cells in situ has suggested to us that IL-1 or TNFct//? do not significantly contribute to the growth of these lesions.^'' As KS lesions express a high level of monocyte chemo-attractant protein 1 (MCP-1)^' which is also found within the basal-cell layer of
tbe epidermis, we presently assume that the growth of KS lesions is a self-amplifying process driven by reciprocal stimulation of KS cells and dermal dendrocytes attracted to the lesions by MCP-1.^' Returning to the tat-protein which has been shown by Ensoli et alP to represent an important growth factor for certain KS cell lines, there are a number of experiments and observations that permit seme fascinating speculation. Vogel et al?^ were able to show that HlV-tattransgenic mice develop tumours which clinically and histologically are similar to KS lesions in man. It is interesting that transgenic mice of both sexes express tatmRNA in the skin but KS-like lesions occur only in male animals which is analogous to the situation of AIDSassociated KS. In addition, it was shown, that tat-mRNA is not expressed in cell lines derived from these tumours. Expanding on these experiments Ensoli et alP provided evidence that tat protein is released by HIV-1 infected H9-cells and tat-transfected COS-1 cells and that not only supernatants containing tat-protein, but also recombinant tat-protein, support the growth of AIDS-KS cells in vitro. Furthermore, this propagation could be inhibited by anti-tat-antibodies. These observations appear to suggest that a viral regulatory gene product is released as a biologically active protein which can serve as a stimulator of cell growth in its own right.^-^ If we now interpret these findings to fit the scenario in HIV-infected patients it will be remembered that AIDSKS lesions contain CD4+ cells^** that are potentially HIVinfected and therefore could release tat-protein which in turn could stimulate the growth of KS lesions. We also know that in HIV-positive patients replication of HIV occurs in epidermal Langerhans cells^" which therefore also could represent a source for tat protein-mediated growth stimulation of KS. As KS lesions arise in the subepidermal dermis it does not appear unreasonable to speculate that signals for KS proliferation could be derived from HIV-infected epidermal Langerhans cells. What is the aetiological agent of KS? A viral aetiology of KS has been discussed for years and the potential role ofthe tat-gene in the growth promotion of this tumour focuses new interest on a possible viral aetiology. In the past, CMV has been discussed as a potential aetiological factor but evidence to support this contention has not been forthcoming.^'^' In the early years of the HIV epidemic KS was also aetiologically linked to HIV, particularly in view ofthe fact that the risk of developing Kaposi's sarcoma is approximately 20000 times greater in AIDS patients than in the general population and approximately 300 times greater than in other types of immunosuppression.^- On the other hand, HIV genes or gene products havenot been detected in KS lesions and there is no correlation hetween the appearance
THE ENIGMA OF KAPOSI'S SARCOMA and course of KS and the degree of immunosuppression.** Further features that argue against a causal relationship between HIV and KS are the observation that more than 90% of KS in HIV-infected individuals occur in male homosexuals and that most HIV-infected individuals do not develop KS; KS also occurs in HIV-negative male homosexuals and women who only rarely have KS, develop this tumour when their HIV-positive partners are bisexual or i.v. drug users. It is also quite striking that the incidence of KS in HIV-positive homosexuals has fallen from 42% in 1983 to 13% in 1988 despite the fact that the incidence of HIV infection has increased.'^ These and other additional reasons have suggested to Beral et al.^^ that KS in AIDS patients is possibly the result of the infection with another sexually transmissible agent that may be concomitantly transmitted with HIV. Indeed, electron microscope investigations have shown that KS cells in AIDS-associated KS^-* ^'' contain intracellular
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inclusions—tubuloreticular structures (TRS) and cylindrical-confronting cisternae (CCC)—which today are interpreted as cellular reaction products to viral infections.^* TRS and CCC are also abundantly present in KS cells of the endemic Mediterranean KS variant'" and classical KS. They can be induced in vitro by interferon (x/^^*'"-'^and this raises the question whether KS could be due to a hitherto unknown retroviral agent which is different from HIV. We have recently found, by electronmicroscopy, virus particles in KS lesions'" that morphologically unequivocally correspond to oncovirinae.^^ These particles occur both as immature and mature viral structures intra- and extracellularly in KS lesions (Fig. 1) and there is morphological evidence suggesting an uptake of such particles via coated pits and their segregation through a process of budding into intracellular vacuoles (Fig. 1). Although so far we have no molecular biological and biochemical evidence to prove the presence of
Figure 1. Retrovirat particles identitied in KS lesions, (a) immature virus with 7-10 mm spikes al the enveloping membrane (arrows) (x 120 000); (h) extracellular virus particte (arrow) close to a KS cell (x 60 000); (c) uptake of retrovirus (arrow) hy coated pit {bold arrow) ( X 60 000); (d) particte (arrow) within a cytoptasmic vacuote of a KS cetl ( x 66 000). (Reproduced from Rappersberger el al.'^\ Journal ofInvestigative Dermatology., with kind permission, Elsevier Science Pubtishing Co. Inc NewYork,)
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retroviruses in KS lesions, the morphological data leave no doubt that retroviral particles do occur on KS. These particles have so far been identified reproducibly in endemic Mediterranean"* and classical KS (unpublished results) so that the retroviruses originally found in HIVassociated KS—originally interpreted to represent HIV*^'*^—could now possibly be interpreted in a different light. Proof of the pathogenetic relevance of these viruses would support the hypothesis of Dictor and Jarplid''-^ which considers KS to be a human retroviral analogue of fowl haemangiomatosis which is caused by a retrovirus ofthe lymphoid leukosis group. Outlook The concept of Kaposi's sarcoma as a retroviral process is indeed fascinating. However, I wish to emphasize that evidence available to date is entirely based on morphological data and merely indicates that retroviral particles are present in KS lesions. We have so far been unsuccessful in characterizing this retrovirus by molecular and biochemical methods and the decisive question therefore remains whether these particles have any pathogenetic significance. Proof of this would however permit us to paint a scenario in which such viruses are transmitted either sexually or non-sexually and in the case of AIDS together with, or independently of, HIV and to initiate cellular processes that directly lead to a transformation and the proliferation of endothelial cells. Indeed it has been shown by Delli-Bovi and Basilico'*'' and by Lo and Liotta^^ that KS-DNA transforms NIH/3T3 cells and that such transformants produce angiosarcomatous lesions in nude mice. Another role of KS retroviruses could consist of the stimulation of the production and secretion of angiogenic and growth factors by endothelial cells and dermal dendritic cells which in turn would initiate a self perpetuating cycle of cellular proliferation by paracrine or autocrine secretion. These lines of thought are presently entirely speculative in nature but they do provide an interesting model for further research.
Acknowledgments I would like to thank the following who have, through their research, made important contributions to this oration: Professor Georg Stingl, Dr Klemens Rappersberger, Dr Reinhard Gillitzer, Dr Rudolf Berger, Dr Erwin Tschachler, Dr Eva Zonzits, Dr Angelos Hatzakis, Dr Dean L.Mann, Dr Robert J.Biggar. References t, Kaposi M. Idiopathisches rnultiptes pigmentsarkom der haut. Archives of Dermatology and Syphilis (Prag) t872; 4: 265-273. 2. Stavin G, Cameron HMcD, Singh H, Kaposi's sarcoma in
mainland Tanzania: a report of 117 cases, British Journal of Camer 1969; 23: 349-357, 3. Taylor JF, Templeton AC, Vogel CL, Ziegter JL, Kyalwazi SK. Kaposi's sarcoma in Uganda: a clinico-pathological study. International Journal oj'Cancer 1971; 8: 122-135. 4. Penn I. Kaposi's sarcoma in organ transplant recipients: report of 20 cases. Transplantation 1979; 27: 8-11. 5. Shumeli D, Shapira Z, Yussim A, Nakache R, Ram Z, Shaharabani E. The incidence of Kaposi sarcoma in renal transplant patients and its relation to immunosuppression. Transplant Proceedings 1989; 21: 3209-3210. 6. Hymes KB, Cheung T, Greene JB, Prose NS, Marcus A, Ballard H, William DC, Laubenstein LJ. Kaposi's sarcoma in homosexual men—report of eight cases. Eancet 1981; ii: 598-600. 7. Friedman-Kien AE, Lauhenstein LJ, Ruhinstein P, BuimoviciKlein E, Marmor M, Stahl R, Spigland I, Kim KS, Zolla-Pazner S. Disseminated Kaposi's sarcoma in homosexual men. Annals of Internal Medicine 1982; 96: 693-700. 8. Safai B. Kaposi's sarcoma: an overview of classical and epidemic (brms. In: Broder S etl, AIDS. Modern Concepts and Therapeuti Challenges. Marcel Dekker, Inc.: New York and Basel, 1987: 205218, 9. Ziegler JL, Dorfman RF (eds), Kaposi's Sarcoma. Pathophysiology and Clinical Management. Marcel Dekker, Inc.: New York and Basel, 1988, 10. Rappersberger K, Tschachler E, Zonzits E, Gillitzer R, Hatzakis A, Kaloterakis A, Mann DL, Popow-Kraupp T, Biggar J, Berger R, Stratigos J, Wolff K, Stingl G. Endemic Kaposi's sarcoma in human immunodeficiency virus type 1-sero-negative persons: demonstration of retrovirus-like particles in cutaneous lesions. Journal of Investigative Dermatology 1990; 95: 371-381. 11. Costa J, Rabson AS. Generalized Kaposi's sarcoma is not a neoplasm, Eancet 1983; i: 58. 12. Brooks JJ. Kaposi's sarcoma: a reversible hyperplasia. Lancet 1986; ii: 1309-1311. 13. NickolofF BJ, Griffiths CEM. Factor Xllla-cxpressing dermal dendrocytes in AIDS-associated cutaneous Kaposi's sarcomas. Science 1989; 243: 1736-1737. 14. Scully PA, Steinman HK, Kennedy C, Truehlood K, Frisman DM, Voland JR. AIDS-related Kaposi's sarcoma displays differential expression of endothelial surface antigens. American Journal of Pathology 1988; 130: 244 251. 15. Green TL, Meyer JR, Daniels TE, Greenspan JS, de Souza Y, Kratner RH, Kaposi's sarcoma in AIDS: hasement membrane and endothelial cell markers in late stage lesions. Journal of Oral Pathology 1988; 17: 266 272. 16. Nadimi H, Saatee S, Armin A, Toto PD. Expression of endothelial cell markers PAL-E and EN-4 and Ia antigens in K.zpQ%i'^ ^'urcomi. Journal of Oral Pathotogy 1988; 17:416-420 17. McNutt NS, Fletcher V, Conant MA. Early lesions of Kaposi's sarcoma in homosexual men. An ultrastructural comparison with other vascular proliferations in skin. American Journal of Pathology 1983; 111:62-77. 18. KrafFert CA, Planus L, Penneys NS. Kaposi's sarcoma: further immunohistologic evidence of a vascular endothelial origin. Journal of Investigative Dermatotogy 1991; 96: 598, 19. Nakamura S, Salahuddin SZ, Biherfeld P, Ensoli B, xMarkham PD, Wong-Staal F, Gallo RC, Kaposi's sarcoma cells: long-term culture with growth factor from retrovirus-infected CD4"'^ cells. Science 1988; 242: 426-430. 20. Salahuddin SZ, Nakamura S, Biberfeld P, Kaplan MH, Markham PD, Larsson L, Gallo RC. Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro. Science 1988; 242: 430-433. 21. Ensoli B, Nakamura S, Salahuddin SZ, Biberfeld P, Larsson L, Beaver B, Wong-Staal F, Gallo RC. AIDS-Kaposi's sarcoma-
THE ENIGMA OF KAPOSI'S SARCOMA
22.
23.
24. 25.
26.
27.
28. 29.
30.
31. 32.
derived cells express cytokines with autocrine and paracrine growth effects. Science 1989; 243: 223-226, Miles SA, Rczai AR, Salazar-Gonzalez JF, Van der Meyden M, Stevens RH, Logan DxVl, Mitsuyasu RT, Taga T, Hirano T, Kishimoto T, Martinez-Maza O. AIDS Kaposi sarcoma cells produce and respond to interleukin 6, Proceedings ofthe Nationat Academy of Sciences ILSA 1990; 87: 4068-4072, Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F, Tat-protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients. Nature 1990; 345: 84-86, Berger R, Purtsche M, Tong D, WolfTK, Stingl G. A tibrohlast growth factor-like activity secreted by a dendritic B cell line. Archives of Dermatotogy Research 1991; 283: 63. Schulze-Osthoff K, Goerdt S, Sorg C, Expression of hasic fibrnhlast growth factor (bFGF) in Kaposi's sarcoma: an immunohistologic study. Journal oj'Investigative Dermatological 1990; 95: 238-240. Gillitzer R, Miiller C, Wolff K, Stingl G, Berger R. High in situ expression of IL-6 mRNA in Kaposi's sarcoma: a possihie role in
151
33. Gyorkey F, Sinkovics JG, Gyorkey P, Tubulo-reticular structures in Kaposi's sarcoma. Lancel 1982; ii: 984 985. 34. Sidhu GS, Stahl RE, El-Sadr W, Zolla-Pazner S. Ultrastructural markers of AIDS, Lancet 198.3; i: 990-991. 35. Sidhu GS, Stahl RE, El-Sadr W, Cassai ND, Forrester ExM, Zoila-Pazner S. The acquired immunodeficiency syndrome: an ultrastructural study. Human Pathology 1985; 16: 377-386. 36. I.uu J, Bockus D, Remington F, Bean MA, liammar SP. Tuhuloreticular structures and cylindrical confronting cisrernae: a review. Human Pathology 1989; 20: 617-627. 37. Yoffe B, Petrie BL, Noonan CA, Hollinger FB. In vivo and in vitro ultrastructural alterations induced by human immunodeficiency virus in human lymphoid cells. Eahoratory Investigations 38. Orenstein JM, Preble OT, Kind P, Schulof R. The relationship of serum alpha-interferon and ultrastructural markers in HIVseropositive individuals. Ultrastructural Pathology 1987; 11: 673-679. 39. Gelderhlom HR, Ozel M, Pauli G. Morphogenesis and morphology of HIV structure-function relations. Archives of Vtrotogy 1989; 106: 1-13. oncogenesis. Archives of Dermatological Research 1991; 283: 40. Gyorkey F, Sinkovics JG, Melnick JL, Gyorkey P. Retroviruses 57-58. in Kaposi-sarcoma cells in AIDS. ?^ew Engtand Journal of Gillitzer R, Stingl G, Burg G, Wolff K, Berger R. High levels of Medicine 1984; 311: 1183-1184. mRNA expression of monocyte chemoattractant protein-1 41. Schenk P, Konrad K. Ultrastructure of Kaposi's sarcoma in (MCP-l) and interleukin 6 (IL-6) in Kaposi's (KS) sarcoma acquired immune deficiency syndrome (AIDS). Archives of lesions in situ. Journal of Investigative Dermatotogy 1991; 96: 598, Otorhinotaryngology 1985; 242: 305-313. Vogel J, Hinrichs SH, Reynolds'RK, Luciw PA, Jay G, The HIV 42. Schenk P. Retroviruses in Kaposi's sarcoma in acquired immune tat gene induces dermal lesions resembling Kaposi's sarcoma in deficiency syndrome (AIDS). Acta Otolaryngology 1986; 10!: transgenic mice. Nature 1988; 335: 606-611, 295-298." Gillitzer R, Berger R, Rappersberger K, Wolff' K, Stingl G, 43. Dictor M, Jarplid B, The cause of Kaposi's sarcoma: an avian Characterization of lymphocyte infiltrates in endemic vs epidemic retroviral -innXo^. Journat ofthe American Academy ofDermatology Kaposi's s-ivcom-i. Journat of Investigative Dermatotogy 1989; 92: 1988; 18: 398-402. 434. 44. Delli-Bovi P, Basitico C. Isolation of a rearranged human Tschachler E, Groh V, Popovic M, Mann DL, Konrad K, Safai transforming gene following transfection of Kaposi sarcoma B, Ezon L, di Marzo-Veronese F, Woltl'K, Stingl G. Epidermal DNA. Proceedings of lhe National Academy of Science USA 1987; Langerhans cells—a target for IITLV-III/LAV infection. Jour84: 5660-5664. nat of Investigative Dermatology 1987; 88: 233-237, 45. Lo SC, Liotta LA. Vascular tumors produced by NIH/3T3 cells Rudlingcr R, Neumann-Haefelin D. Zytomcgalieviren und transfected with human AIDS Kaposi's sarcoma DNA. American Kaposi-Sarkom, Hautarzt 1987; 38: 573-576. Journal of Pathology 1985; 118: 7-13. Beral V, Peterman TA, Berkelman RL, Jaffe HW, Kaposi's sarcoma among persons with AIDS: a sexually transmitted infection? Lancet 1990; 335: 123-128.
