Oldest known case of metastasizing prostate carcinoma diagnosed in the skeleton

of a 2,700-year-old Scythian King from Arzhan (Siberia, Russia)

Michael Schultz1*, Hermann Parzinger2, Dmitrij V. Posdnjakov3, Tatjana A. Chikisheva3 and Tyede H. Schmidt-Schultz4

1Department of Anatomy, University of G€ottingen, Germany2German Archaeological Institute (DAI), Berlin, Germany3Institute of Archaeology, Russian Academy of Sciences, Novosibirsk, Russia4Department of Biochemistry, University of G€ottingen, Germany

To determine whether a 2,700-year-old tumor can be reliably diag-nosed using microscopic and proteomic techniques and whethersuch prostate carcinomas show the same morphological pattern atthe micro-level as modern-day carcinomas, this case was investi-gated. A 40–50-year-old Scythian king who lived during the IronAge in the steppe of Southern Siberia (Russia) suffered from mac-roscopically visible osteoblastic and osteoclastic lesions throughouthis entire skeleton. Macro-morphological (macroscopy, endoscopy,radiology) and micro-morphological techniques (histology, scan-ning-electron microscopy) as well as proteomic techniques (1-D-and 2-D-electrophoresesis, Western blot) were applied. The resultsof the morphological and biochemical investigation proved thatthis mature male suffered for many years from and probably diedof a carcinoma of the prostate. The diagnosis mainly rests on theresults of the microscopic examination of the lesions and the posi-tive evidence of PSA, which is an important marker for the diagno-sis of prostate cancer. It is remarkable that, in this ancient case,the morphological pattern at the microlevel is the same as in recentcases. The loss of the spongy bone substance (red bone marrow)provoked chronic anemia during the final months of the life of thisking. The proteomic techniques applied are new for the investiga-tion of recent and ancient macerated bones. Sensitive and reliablebiochemical markers (PSA) are an important precondition todetect such tumors in recent and ancient materials. Currently, thisis the oldest known case of prostate cancer diagnosed reliably bymorphological and biochemical techniques.' 2007 Wiley-Liss, Inc.

Key words: extracellular bone matrix proteins; history of tumorousdiseases; paleopathology; prostate carcinoma; proteomics

It is our goal to obtain certain information on ancient diseasesfrom the dawn of civilization using archeological skeletal remainsas bio-historical documents because the knowledge of the nature,etiology and epidemiology of ancient diseases might provideinsights into the future health problems. This skeletal investigationwas part of the interdisciplinary study ‘‘Health and Disease inEarly Nomadic Populations from Asia’’ directed by the G€ottingenUniversity and the German Archaeological Institute at Berlin.

The paleoproteomic investigation of intact extracellular matrix(ECM) proteins of bone and teeth extracted from human beingswho lived thousands of years ago is a new field in biochemistry.The molecules of cells (DNA, proteins, lipids) degrade after death,however, the ECM-proteins that are situated outside cells andbound to the ossified connective tissue remain protected by thescaffold of hydroxyapatite over thousands of years.1

It is remarkable that it is at all possible to obtain informationfrom DNA extracted from ancient bones and teeth, which areseverely degraded and this information is indeed extremely lim-ited. Additionally, in ancient DNA, there is a high risk of contami-nation by foreign DNA. In ECM-proteins, however, there isalmost no contamination risk because these are bound to the hy-droxyapatite scaffold as in a living organism. The only real prob-lem is to bring these intact proteins into solution. With ourmethod, we can solubilize all the intact ECM-proteins from recentand ancient bones and teeth with the same quality.2,3 This is theprecondition to establish reliable disease diagnoses also at themolecular level using ECM-proteins extracted from individualsdating from ancient times. Indeed, proteins are the real player in

every step of metabolism and now we also have a tool to establisha reliable diagnosis at the level of intact proteins from the Scy-thian ruler of Arzhan, who lived 2,700 years ago.

Material and methods

Materials

The relatively well-preserved skeleton of a 40–50-year-oldunknown Scythian king excavated over a period between 2000and 2003 from Kurgan 2, burial 5 at Arzhan near Tuva (southernSiberia, Russia), and dating from the 7th century BC represents anundisturbed burial, which is one of the richest graves from theEurasian Steppes and contained about 9,600 objects, more than6,000 made of gold.4

Samples for the microscopic and biochemical analysis are asfollows: (i) Arzhan Kurgan 2, burial 5, individual 1, male, 40–50-year-old. For light and scanning-electron microscopy, sampleswere taken from the right parietal bone, the right humerus, theright femur, the body of the seventh thoracic vertebra and the leftfourth and fifth rib. For biochemistry, a sample was taken from theleft tibia and the left fifth rib, (ii) recent male, 62-year old (Depart-ment of Anatomy, University of G€ottingen, Germany), sufferingfrom prostate cancer. For biochemistry and light microscopy, asample was taken from the right femur, (iii) recent male, 74-yearold (Department of Anatomy, University of G€ottingen, Germany),no systemic disease known. For biochemistry and light micros-copy, a sample was taken from the right femur.

Morphological techniques

The skeleton was examined using macroscopic, endoscopic, ra-diological, light microscopic and scanning-electron microscopictechniques.5 For histology, resin-embedded thin ground sectionswere viewed in polarized transmission light.5

Biochemical techniques

For the first time in paleopathology, a new biochemical tech-nique has been applied.1 Molecular paleopathology is a new fieldof research in which molecules are used to confirm the presence ofdisease in past populations.2 The human bone ECM-proteins wereextracted and solubilized, resolved by SDS-PAGE and 2-D-elec-trophoresis and identified in Western blot.

Protein extraction

During the last 25 years, ECM-proteins from bone were usuallyextracted using a technique that was developed by Termine et al.6

They extracted the proteins from the supernatants after the centrif-ugation steps.6 We have changed this method in a decisive pointin that we always work with the pellets. The supernatants were

*Correspondence to: Department of Anatomy, University ofG€ottingen, Kreuzbergring 36, D-37075 G€ottingen, Germany.Fax:149-551-39-7043. E-mail: mschult1@gwdg.deReceived 1 June 2007; Accepted after revision 6 June 2007DOI 10.1002/ijc.23073Published online 4 October 2007 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 121, 2591–2595 (2007)' 2007 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

discarded, and we also solubilized the ECM proteins from the lastlyophilized pellet.1,2

1-D- and 2-D-electrophoresesis

The solubilized proteins were precipitated with trichloraceticacid and separated by SDS-PAGE (1-D7) or by 2-D-electrophore-sis. The dimension in 2-D-electrophoresis is carried out with animmobilized pH gradient (IPG) 7-cm strip, pH 3–10. Focusingwas performed at a maximum of 20�C (Protean IEF-cell, Bio-Rad,CA), 4,000 and 10,000 V h. The equilibrated IPG strip8 was trans-ferred to a polyacrylamide gel (11% T, 2.5% C) without stackinggel. The gels were stained with silver according to a commonlyused protocol.9

Western-blot analysis

The proteins were identified by Western blot with specific anti-bodies. Nondenatured and nonreduced protein samples (30 lg)were separated by SDS-PAGE with 11% total acrylamide (T) and2.5% cross-linker (C). The ECM proteins of human bone weretransferred in a Trans-Blot cell (Bio-Rad, CA) with water coolingfor the temperature control to polyvinyl dine fluoride (PVDF)membranes in a CAPS (3-cyclohexylamino-1-propane-sulfonicacid) buffer, pH 11. The nonspecific-binding sites on the PVDFmembrane were blocked with 1% BSA (bovine serum albumin).The transferred proteins were incubated with MAB antihuman-PSA (diluted 1:100; Chemicon, Harrow, UK) or MAB anti-PSA-ACT (diluted 1:1,000; Upstate, Lake Placid, NY). Horseradishperoxidase-linked anti-mouse (diluted 1:5,000; Bio-Rad, CA) wasused as secondary antibody. Bands were visualized using anenhanced chemiluminescence (ECL)-Plus detection system.

Results

The morphology of the tumorous lesions

This ruler from Arzhan suffered from macroscopically visibleosteoblastic and osteoclastic lesions throughout his entire skele-

ton. Almost all bones were affected by the disease process mainlyexpressed by porotic, coarse and platelike lesions (Fig. 1a). Thelight microscopic investigation revealed almost no original bonestructure. There were pronounced vestiges of an irregular rapidbone growth characterized by a primary osteoclastic (Fig. 1b) anda secondary dominating osteoblastic process (Fig. 1c). In the cra-nial sample, the original bone had been replaced by a bulky,almost compact structure causing a loss of the diploic modules ofthe red bone marrow. The loss of the spongy bone substance,which represents the modules of the red bone marrow, causedchronic anemia during the final months of the king’s life. Thecompact bone substance of the long bones had been largelydestroyed by osteoclastic resorption (Fig. 1c). Remaining parts ofthe compact bone substance had been replaced by secondarilybuilt primitive woven bone. Very similar changes were observedin the samples taken from ribs and vertebrae. The nature, speedand direction of the growth of the newly built bone formations(e.g., trabeculae, subperiosteal bone apposition) as well as the typ-ical resorption holes (Fig. 1c) characterized the disease as a nonin-fectious and tumorous process. The morphology and the pattern ofthe lesions are the characteristic of the carcinoma of the prostate.

Ancient human ECM proteins separated by 1-Dand 2-D-electrophoresis

ECM proteins were extracted from the samples of human com-pact (cortical) bone substance,1,2 taken from the ruler from Arzhanand two recent individuals. The silver-stained 1-D-electrophoresis(SDS-PAGE) shows the individual from Arzhan (Fig. 2a, lane 1,ECM proteins from the left tibia, lane 2, ECM proteins from thefifth left rib) with a protein pattern of �25–30 colored bands witha molecular mass range of �10–200 kDa. Separation by SDS-PAGE yields many proteins with the same molecular weight. Tocalculate the quantity of proteins in the bone ECM, it is necessaryto separate the proteins by 2-D-electrophoresis. The 2-D-electro-phoresis shows more than 300 silver-stained protein spots (Fig. 2b)in the molecular weight range of 10–200 kDa and with isoelectric

FIGURE 1 – Lesions in right humerusof the king from Arzhan. (a) Macro-scopic view. The shaft, whose surface ischaracterized by secondarily built boneplates, is clearly thickened in its proxi-mal part due to tumorous growth. (b)Scanning-electron microscopic image.Smooth and porous bony plates on theexternal surface of the shaft of right hu-merus which are a characteristic featureof tumor periostosis. Scale of bar is 100lm. (c) Cross section through shaft ofright humerus. Bony plates on the exter-nal surface of shaft. The compact bonesubstance is severely damaged byresorption holes due to osteoclastic ac-tivity. Microphotograph of thin groundsection (thickness 50 lm) viewed inpolarized light using a hilfsobject red 1storder (quartz) as compensator. Scale ofbar 200 lm.

2592 SCHULTZ ET AL.

points between pH 3 and pH 10. This is the range that includesmost of the proteins in a living organism.

PSA identified in a 2,700-year-old human skeleton

Monoclonal antibodies against human PSA show the positivebands in a Western blot with a molecular weight of about 75 kDa(Fig. 3a), lane 1 is the result from the ruler of Arzhan and lane 2from the recent individual who was suffering from prostate cancer,no bands were identified in the recent individual not sufferingfrom prostate cancer (Fig. 3a, lane 3). The PSA antigen in humanserum exists as free PSA and as complexed PSA with a1-antichy-motrypsin (PSA/ACT, about 75 kDa).10 We tested whether PSA inthe human ECM is complexed to ACT. The monoclonal antibodyagainst human PSA/ACT in the Western blot also showed a posi-tive band of 75 kDa in the bone from the ruler of Arzhan (Fig. 3b,lane 1) and from the recent individual with prostate cancer(Fig. 3b, lane 2). The recent individual not suffering from prostatecancer shows no bands (Fig. 3b, lane 3).

Discussion

In the recent cases of prostate cancer, the development of char-acteristic skeletal metastases of more osteoblastic than osteoclastic

activity occurs in 70% of patients.11 There is no reliable informa-tion at the microscopic or proteomic levels on the nature, occur-rence, and mode of metastases of ancient tumors, which would beuseful for the understanding of the history and evolution of tumor-ous diseases. However, such investigations are interesting becausecertain types of cancer that these days are estimated to be charac-teristic of our own times and frequently described as being due toour western civilization are also found in antiquity.

Special diagnostic criteria have to be used to establish diagno-ses in archaeological skeletal remains because of the loss of cellsand soft tissue structures.5,12 Local spreading of systemic boneresorption products of malignant tumors leads to bone destruction.Thus, in general, cancer of the prostate gland, lung, breast, kidney,stomach, thyroid gland as well as multiple myeloma frequentlymetastasize in the skeleton.13–15 Additionally, carcinomas arisingin other organs, such as uterus, ovary, suprarenal gland, bladderand testicle might metastasize in a similar way; however, they rel-atively seldom affect the bony system. A malignant tumor candestroy the bone mass by local invasive growth or by producingenzymes with an ability to resorb systemic bone.16–18 Usually, car-cinoma of the prostate gives rise to the solidification or condensa-tion of bone. However, sometimes, in individual cases, also carci-nomas of the breast or the stomach give rise to formative or osteo-blastic metastases, whereas a carcinoma of the prostate is, in

FIGURE 2 – 1-D- and 2-D-elec-trophoresis of the ECM-proteinsof compact bone from the rulerof Arzhan. (a) ECM-proteins wereresolved by 1D-electrophoresis(SDS-PAGE) and silver stained,lane 1 ECM-proteins from lefttibia, lane 2 ECM-proteins fromleft fifth rib. (b) 2-D-electrophore-sis resolved proteins according totheir molecular weight (from top tobottom) and their isoelectric point(between pH 3 and pH 10). Theprotein spots (ECM-proteins fromleft tibia) were silver stained.

FIGURE 3 – Western blot. (a) Monoclo-nal antibodies against prostate specificantigen (PSA). Lane 1 ECM proteins fromleft tibia of the ruler of Arzhan, lane 2ECM-proteins from right femur of arecent male with known prostate cancer,lane 3 ECM-proteins from right femur ofa recent male not suffering from prostatecancer, on left the molecular weightmarker. (b) Monoclonal antibodies againstPSA complexed with a1-antichymotrypsin(PSA/ACT). Lane 1 ECM-proteins fromleft tibia of the Arzhan ruler, lane 2 ECMproteins from right femur of a recent malewith known prostate cancer, lane 3 ECM-proteins from right femur of a recent malenot suffering from prostate cancer, on theleft molecular weight marker.

2593PCA DIAGNOSED IN THE SKELETON OF A SCYTHIAN KING

addition, able to produce osteoclastic metastases.19 Unlike mostother secondary bone metastases, the majority of skeletal prostatemetastases are osteoblastic rather than osteolytic in nature.11,20

Thus, the macroscopic and the microscopic features of the lesionsobservable in the skeleton of the king from Arzhan are characteris-tic of prostate cancer. As a rule, metastases occur through bloodvessels, lymphatic vessels and directly by the extensional growthof the primary tumor or from other, already established metastases.In the case of the ruler from Arzhan, blood vessels and/or lym-phatic vessels probably spread the metastases. However, a meta-stasizing process along the perineural lymphatics, which is fre-quently seen in the carcinoma of the prostate, cannot be excluded.

In comparison with the recent cases, the micro-morphologyof the product of the osteoblastic processes in the skeleton fromArzhan is, indeed, the same. Up to now, the micro-morphology ofprostate cancer has not been investigated in macerated bone speci-mens with our techniques. Using thin ground sections examinedin plane and polarized transmission light microscopy and scan-ning-electron microscopy, typical morphological features charac-teristic of different diseases are diagnosable.5,12

It is now possible to solubilize the whole set of ECM proteins inrecent and ancient bone. After being resolved by 2-D-electropho-resis, more than 300 different protein spots are stained with silver.Several of the extracted proteins represent isoenzymes or have dif-ferent glycosylation patterns, so that we can probably solubilizemost, if not all, ECM proteins, which are present in compact bone.It is assumed that �200 different matrix proteins are present inbone.21

The results show that certain molecules, which were extractedfrom diseased cells typical for this disease, were transported bythe blood and absorbed to bone hydroxyapatite. Prostate-specificantigen (PSA) is an �30 kDa secretory protein of the human pros-tate.22 By mechanisms, which have, up to now, remainedunknown, a small amount of PSA is released from the prostateinto the blood circulation.23 The exact mechanism on how PSAends up in blood serum and then also in the ECM of bone is notexactly understood but the leakage is promoted by tissue abnor-malities within the prostate that allow the enzyme to travelthrough the cell stoma into the bloodstream via capillaries andlymphatic pathways. PSA exists in more than one form circulatingin the serum.10 These forms are (i) free PSA and (ii) bound PSA,which is complexed with 1-antichymotrypsin (PSA-ACT). The

ratio-free total PSA (free PSA and bound PSA) in serum has beendemonstrated to significantly improve the discrimination of pros-tate cancer from benign prostate hyperplasia (BPH) with higherlevels of free PSA correlating with a lower risk.24,25 In the case ofprostate malignancy, the concentration of bound PSA in-creases.26,27 Results of these studies demonstrate that most of theimmunologically detectable PSA in the ECM of bone is com-plexed with ACT. Free PSA was not detectable in the bone sam-ples of the king from Arzhan. ACT is a glycoprotein with a massof between 50 and 65 kDa. The variation in size is attributed to avariation in glycosylation.28 Thus, our biochemical result demon-strates that the tumor was in a state of high malignancy.

The results show that the molecules, typical of a special disease,were transported by the blood and absorbed to bone hydroxyapatiteduring lifetime. In the skeleton of the Arzhan King, we can identifythese molecules in the bone ECM even thousands of years after hisdeath. High-PSA levels were detected in a well-preserved bonesample, and the ECM proteins are bound to apatite just as in livingbone. They were not degraded after death as were the cell mole-cules (e.g., DNA, other proteins). Therefore, it is now possible toconfirm the diagnosis of tumorous diseases by specific tumormarkers also in ancient specimens. The proteomic techniques usedare new for the study of ECM proteins extracted from recent andancient macerated bone samples. This method is a precondition forthe detection of tumor markers in macerated bones, such as PSA.

This observation provides, for the first time, key insights into areliable diagnosis of prostate carcinoma in ancient times, whichgive new impulse to models for the further studies in the field ofthe comparative evolution and the history of tumorous diseasesstarting from ancient and leading up to modern times.

Acknowledgements

The authors gratefully acknowledge the generous support ofIrmelin Probst, Department of Biochemistry, University ofG€ottingen. Furthermore, they thank Michael Brandt, IngridHettwer-Steeger and Hannes Sydow for technical assistance,Cyrilla Maelicke for reading the English text and all from theDepartment of Anatomy, University of G€ottingen. Mr. MichaelSchultz received funding from the University of G€ottingen, Mr.Hermann Parzinger from the German Archaeological Institute andthe German National Sciences Foundation.

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