Gino Fornaciari, Silvia Pellegrini, Generoso Bevilacqua, Antonio Marchetti
Molecular Pathology and *Paleopathology Sections
Institute of Pathology, Medical School
University of Pisa, 56126, Pisa, Italy.

The autopsy performed on the artificial mummy of Ferrante I of Aragon, King of Naples and leading figure of the Italian Renaissance (1431-1494), exhumed in the Abbey of Saint Domenico Maggiore, revealed a tumor infiltrating the small pelvis.
Histology showed exceptionally preserved neoplastic cells, disposed in cords and solid nests or forming pseudo-glandular lumina, disseminated in a fibrous tissue containing striated muscular fibres.
These findings clearly indicated an infiltrating epithelial malignant tumor, probably a colon or prostatic adenocarcinoma.1 

Since colorectal tumors are characterised by frequent mutations of the K-ras oncogene, whereas in prostatic adenocarcinomas such mutations are extremely rare, we decided to investigate the status of the K-ras gene in tumor DNA extracted from the mummified tumor tissue.

Paraffin blocks containing predominantly neoplastic cells were chosen for analysis. Five sections (10 mm thick) were collected in an Eppendorf tube and processed for DNA extraction as previously described.2 
Genomic DNA obtained from the skin of the same mummy and pure water were used as controls. The samples were subjected to a nested PCR protocol designed to yield a 77 bp K-ras fragment encompassing codon 12, the main hotspot for mutations in colon cancer. The amplified products of the PCR reaction were denatured and blotted onto nylon membranes which were then hybridized separately with 32P-labelled mutation specific oligonucleotide probes.3
A K-ras codon 12 point mutation was present in the tumor sample: the normal sequence GGT (glycine) was altered to GAT (aspartic acid).
The analysis was repeated several times with constant results.
These data strongly suggest that Ferrante I of Aragon was affected by a cancer of the digestive tract, most probably a colon adenocarcinoma.

The observed genetic change indeed represents the most frequent mutation of the K-ras gene in present-day colorectal cancer and it is thought to be induced by alkylating agents.4 
Therefore we speculate that mutagens similar to those responsible for the induction of K-ras mutations in contemporary colon cancer could be present in the rich diet of a court of XV century, as supported by paleonutritional studies.5 

To the best of our knowledge, this is the first time an oncogene mutation has been evidenced in an ancient tumor.

1 Fornaciari G, Castagna M, Naccarato AG, Collecchi P, Tognetti A, Bevilacqua G. Adenocarcinoma in the mummy of Ferrante I of Aragon, King of Naples (1431-1494). Paleopathol Newsl 1993; 82: 7-11.
2 Marchetti A, Merlo G, Buttitta F, Pellegrini S, Callahan R, Bistocchi M, Squartini F. Detection of DNA mutations in acid formaline fixed-paraffin embedded specimens by polymerase chain reaction-single strand conformation polymorphysm analysis. Cancer Det. Prev 1995;19: 278-281.
3 Verlaan de Vries M, Bogaard ME, van den Elst H, van Boom JH, van der Eb AJ, Bos JL. A dot-blot screening procedure for mutated ras genes using synthetic oligodeoxynucleotides. Gene 1986; 50: 313-320.
4 Topal MD. DNA repair, oncogenes and carcinogenesis. Carcinogenesis 1988; 9: 691-696.
5 Fornaciari G, Ceccanti B, Corcione N, Bruno J. Recherches paléonutritionelles sur un échantillon d’ une classe socialement élevée de la Renaissance italienne: la série de momies de S. Domenico Maggiore à Naples (XV-XVI siècles). In: Advances in Paleopathology, Proceedings of the VII European Meeting of the Paleopathology association (Lyon, September 1988), 1989: 81-84.