International Journal of OsteoarchaeologyInt. J. Osteoarchaeol. 9: 302–306 (1999)

K-ras Mutation in the Tumour of KingFerrante I of Aragon (1431–1494) andEnvironmental Mutagens at theAragonese Court of NaplesGINO FORNACIARI*, ANTONIO MARCHETTI, SILVIA PELLEGRINI ANDROSALBA CIRANNIDepartment of Oncology, Division of Pathology, Section of Palaeopathology,University of Pisa, Pisa, Italy

ABSTRACT The autopsy of the mummy of Ferrante I of Aragon, King of Naples (1431–1494) revealed anadenocarcinoma extensively infiltrating the muscles of the small pelvis. A molecular study ofneoplastic tissue observed a typical mutation of the K-ras gene codon 12: the normal sequenceGGT (glycine) was altered into GAT (aspartic acid). The recorded genetic change represents themost frequent mutation of the K-ras gene in sporadic colorectal cancer and is characteristic ofthe effects of alkylating agents. Recent studies have focused the importance of some alkylatingagents, such as the endogenous N-nitroso compounds (NOC). Increased intake of red meat,such as beef, lamb or pork, induces a significant threefold increase of faecal NOC levels, with arange of exposure in faeces similar to that from tobacco-specific NOC in cigarette smoke. Thestudy of alimentary regimens of the Italian Renaissance courts, and in particular of the Aragonesecourt of Naples, produced evidence of very high red meat consumption, attested also by thepalaeonutritional data. Therefore, the alimentary ‘environment’ of the Neapolitan court of the 15thcentury, with the abundance of natural endogenous alkylating agents, can well explain the K-rasmutation causing the tumour that killed the Aragonese king over five centuries ago. Copyright© 1999 John Wiley & Sons, Ltd.

Key words: mummy; adenocarcinoma; DNA; oncogenesis; NOC; Renaissance

Introduction

The autopsy of the mummy of Ferrante I ofAragon, King of Naples and a leading figure ofthe Italian Renaissance, who died in 1494 at theage of 63 years (Figure 1), revealed a moder-ately differentiated adenocarcinoma of thecolon, extensively infiltrating the muscles of thesmall pelvis. Histology showed exceptionallywell-preserved neoplastic cells, disposed incords and solid nests or forming pseudo-glandu-lar lumina (Figure 2), disseminated in a fibroustissue containing striated muscular fibres. Thecells appeared tall and crowded, with abundant

cytoplasm and quite regular hyperchromatic nu-clei (Fornaciari et al., 1993).

The excellent, almost exceptional, preserva-tion of the histological structure of this uniquetumour is probably due to the very dry microcli-matic conditions of the church, and certainly tothe preserving substances used in embalming(Fornaciari, 1998).

As described in an ancient handbook on em-balming (Lanzoni, 1693), in Naples the bodieswere washed with large quantities of aqua vitaeand lye after being eviscerated; this treatment islikely to have ‘fixed’ the tissues, thus protectingtheir histological integrity. Furthermore, abun-dant vegetable resins poured into the cavities ofthe bodies, perfectly preserved the residualorgans.

* Correspondence to: Department of Oncology, University ofPisa, Via Roma, 57-56126 Pisa, Italy. Tel.: +390 50 992894;e-mail: g.fornaciari@do.med.unipi.it

CCC 1047–482X/99/050302–05$17.50Copyright © 1999 John Wiley & Sons, Ltd.

Received 4 December 1998Accepted 26 January 1999

K-ras Mutation 303

Figure 1. Portrait of Ferrante I of Aragon, at about 50 years ofage, revealing severe obesity. From the ‘Adorazione dei Magi’by M. Cardisco. Museum of Capodimonte, Naples.

At present, colorectal cancer is a significantcause of morbidity and mortality in Westernpopulations. Epidemiological evidence suggeststhat elevated meat consumption and/or animalfat intake may increase the risk of the tumour(Payne, 1990; Voskuil et al., 1997). This cancerdevelops as a result of the pathologic transfor-mation of the normal colonic epithelium into aninvasive carcinoma.

It is generally accepted that genetic and envi-ronmental factors combine in the aetiology ofthe tumour. Its multi-step progression takesyears and possibly decades with a number ofrecently characterized genetic alterations (Gryfeet al., 1997).

Among these alterations, a mutation leadingto disregulation of the K-ras protooncogene isthought to be an early event in colon cancerformation. Molecular study by PCR of the neo-plastic tissue in the mummy of Ferrante II evi-denced a typical point mutation of the K-rasgene codon 12: the normal sequence GGT(glycine) was altered into GAT (aspartic acid)(Marchetti et al., 1996).

The recorded genetic change represents themost frequent mutation of the K-ras gene insporadic colorectal cancer and is characteristicof the effects of alkylating agents (Topal, 1988).

Figure 2. Adenocarcinoma with pseudo-glandular lumina in fibrous stroma. H&E, original magnification: ×200.

Copyright © 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 302–306 (1999)

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Figure 3. Possible relation between a diet rich in red meat andthe formation of N-nitroso compounds (NOC).

recent introduction which, in the case of KingFerrante, can be easily excluded.

The N-nitroso compounds (NOC) and theirprecursors, which have been widely investigatedfor their diffusion and well-known relevance ingastrointestinal cancerogenesis, need furtherconsideration. In fact, the carcinogenic proper-ties of N-nitroso compounds are also associatedwith their ability to alkylate DNA (Georgiadis etal., 1991; Mirvish, 1995).

In the case of King Ferrante we can excludesaltpetre, which until some years ago waslargely used as an additive to sausages, as apossible source of nitrites favouring the forma-tion of NOC; the transition from vegetable dyesto saltpetre for the colouring or colour preserva-tion of meat occurred only between 1600 and1750, probably closer to the year 1700 (Lauer,1991).

Recent studies have focused on the impor-tance of the endogenous NOC. As a matter offact, the diet contains a large number of con-stituents that can be nitrosated in the gas-trointestinal tract (especially in the stomach).Therefore, the endogenous NOC burden fromdietary ureas and aromatic amines may representas large a hazard as the intake of pre-formedNOC (Figure 3; Shepard & Lutz, 1989).

In recent years, alkylating agents, because oftheir ability to react directly with DNA either invitro or in vivo, have been used extensively tostudy the mechanisms of mutagenicity and car-cinogenecity. The occurrence of these agents iscurrently widespread in the environment andhuman exposure from natural and pollutantsources is universal (Beranek, 1990).

The alkylating agents can be of natural orartificial origin. The latter includes a large num-ber of chemical and pharmacological substancesof industrial origin, as well as drugs of very

Figure 4. Scene of Banquet at court. From a wedding chest of the 15th century. Riccardiana Library, Florence.

Copyright © 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 302–306 (1999)

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Table 1. Banquet for the wedding of Giangiacomo Trivulzioand Beatrice D’Avalos (Naples, 22 April 1487) Benporat, 1996

Small twisted pine seed and sugar pies, almond andsugar bunsAsparagusOffalsGiblets, chicken’s liverRoasted and spiced partridgesSkin-covered beef and veal headsCapon, pigeon, salami, ham, vegetable soupRoast wether served in a sour cherry brothTurtledove, partridge, pheasant, quail, thrush, woodcockwith olivesPullet cooked in sugared, rose-scented waterRoast piglet in acid brothRoast peacock with seasoningWhipped eggs, cream, milk, sage, flour and sugarQuinces cooked in sugar, cinnamon, pine seeds,artichokesA variety of sugar and honey cakes

natural endogenous alkylating agents, can wellexplain the K-ras mutation causing the tumourthat killed the Aragonese king over five cen-turies ago.

References

Archi, A. (1968) Gli Aragona di Napoli. Bologna:Capelli Editore.

Benporat, C. (1996) Banchetti e Conviti. In: CucinaItaliana del quattrocento ‘Archivium Romanicum’ Library(edited by L.S. Olschki), Florence, 57–71.

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Bingham, S.A., Pignatelli, B., Pollock, J.R., Ellul, A.,Malaveille, C., Gross, G., Runswick, S., Cum-mings, J.H. and O’Neill, I.K. (1996) Does in-creased endogenous formation of N-nitrosocompounds in the human colon explain the associ-ation between red meat and colon cancer? Carcino-genesis, 17: 515–523.

Fornaciari, G., Ceccanti, B., Corcione, N. and Bruno,J. (1989) Recherches paleonutritionelles sur unechantillon d’une classe socialement elevee de laRenaissance italienne: la serie de momies de S.Domenico Maggiore a Naples (XVe–XVIesiecles). In: Advances in Palaeopathology, Proceedings ofthe VII European Meeting of the Palaeopathology Associa-tion, Lyon, September 1988, 81–87.

Fornaciari, G., Castagna, M., Naccarato, A.G.,Tognetti, A., Collecchi, P. and Bevilacqua, G.(1993) Adenocarcinoma in the mummy of FerranteI of Aragon, King of Naples (1431–1494).Palaeopathology Newsletter, 82: 7–11.

Fornaciari, G. (1998) Italian mummies. In: Mummies,Disease & Ancient Cultures, 2nd edn. (edited by A.Cockburn, E. Cockburn and T.A. Reyman). Cam-bridge: Cambridge University Press, 266–281.

Georgiadis, P., Xu, Y.Z. and Swann, P.F. (1991)Nitrosamine-induced cancer: O4–alkylthymineproduces sites of DNA hyperflexibility. Biochem-istry, 30: 11725–11732.

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Lanzoni, I. (1693) Tractatus de balsamatione cadaverum.Parma: Antonius Carrarae, 1693.

Lauer, K. (1991) The history of nitrite in humannutrition: a contribution from German cookerybooks. Journal of Clinical Epidemiology, 44: 261–264.

Increased intake of red meat induces a signif-icant (threefold) increase of faecal N-nitrosocompound (NOC) levels, with a range of expo-sure in faeces similar to that from tobacco-specific NOC in cigarette smoke (Bingham etal., 1996). This endogenous formation of NOCin the human colon explains the associationbetween red meat and colorectal cancer.

The study of the alimentary regimens of theItalian Renaissance courts (Figure 4), and inparticular of the Aragonese court of Naples(Table 1), evidences a very high consumption ofred meat, also attested by palaeonutritional data(Fornaciari et al., 1989).

With regard to the physio-pathological per-sonality of King Ferrante, the portraits, eithersculptures or pictures, reveal a growing obesityfrom youth to maturity (Figure 1; Archi, 1968).Chroniclers report on the strong preference ofthe sovereign for the red meat, especially forthe game (Vultaggio, C., 1996, Personal com-munication).

Examination of the mummy confirms theobesity, of cushingoid type, and reveals severeatherosclerosis of the carotid arteries. Finally,owing to the very high rate of zinc and lead inthe bones, palaeonutritional data show a mas-sive intake of animal proteins and most proba-bly, of wine (Fornaciari et al., 1989).

Therefore, the alimentary ‘environment’ ofthe Neapolitan court of the 15th century andthe sovereign’s habits, with their abundance of

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Marchetti, A., Pellegrini, S., Bevilacqua, G. and For-naciari, G. (1996) K-ras mutation in the tumor ofFerrante I of Aragon, King of Naples (1431–1494). The Lancet, 347: 1272.

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Copyright © 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 302–306 (1999)