[Macroscopic and endoscopic examinations of the head and neck region in ancient Egyptian mummies]. / Makroskopische und endoskopische Untersuchung der Kopf-Hals-Region an altägyptischen Mumien.

INTRODUCTION: The examination of mummies has mostly been performed by macroscopic investigation after unwrapping. During the last decades, however, several research groups provided clear evidence that the combination of various noninvasive approaches for the examination of mummies offers distinct advantages over the previously used methods of unwrapping and inspection. Particularly, the introduction of endoscopic techniques has been used for a closer examination of mummies without destroying them. METHODS: In the last 5 years we analysed about 250 mummies and skeletons found in the necropolis of Thebes-West, Upper Egypt, with particular reference to normal and pathological findings in the head and neck region. Beside macroscopic examination we used endoscopes for the inspection of the nasal cavity and the ear. RESULTS: Most individuals revealed normal macroscopic and endoscopic features. In particular, several skulls showed the auditory ossicles in normal anatomic position indicating an excellent conservation of the specimens. Nevertheless, pathological alterations could be detected affecting different regions of the head and neck. In particular, several individuals presented with fractures of the nasal bones. One case even revealed a severe old-healed fracture of the mid-face (type Le Fort III) with complete loss of all teeth, suggesting adequate "therapeutic" treatment of the skull fracture. Further findings, evidenced by endoscopy, were dentogenic sinusitis and chronic middle ear infections with intracranial perforation in one case. In addition, in one case fixation of the stapes suggests the residues of subluxation of the stapes. CONCLUSION: In this study, we provide further evidence that a careful macroscopic and endoscopic investigation of mummy skulls reveals important information on the state of conservation of the study population and may unravel distinct paleopathological diseases of the head and neck region.

Divergence in distribution and prognostic significance of major basement components in laryngeal carcinomas.

In previous studies, we demonstrated a loss of major basement membrane (BM) components in laryngeal squamous cell carcinomas and provided initial evidence that this was of potential prognostic significance. In our current study, we extended the panel of BM antibodies and enlarged our study group in order to perform a multivariate statistical analysis. We analyzed 26 laryngeal squamous cell carcinomas immunohistochemically for the distribution of the BM-components collagen IV, collagen VII, laminin-1, laminin-5, perlecan and fibronectin. The resulting data were correlated with clinical prognostic factors and statistical correlation coefficients were determined for independent uni- and multivariate analysis. All carcinomas analyzed revealed defects of the peritumoral BM with more extensive loss of collagen VII than collagen IV, laminin-1, perlecan and fibronectin. Laminin-5 in contrast was present even in poorly differentiated tumors showing an enhanced intracytoplasmatic staining in the tumor cells. Furthermore, our statistical analysis did not show independent prognostic significance of any of the BM-components. Our observations indicate a divergence between the loss of several major BM-components (collagens IV, VII, laminin-1, perlecan) and an enhanced deposition of laminin-5. This suggests a severely altered cell-matrix interaction, since laminin-5 links the collagen VII-containing anchoring fibrils to cell receptors of the integrin type.

Synthesis and degradation of basement membranes and extracellular matrix and their regulation by TGF-beta in invasive carcinomas (Review).

The proper structure of the extracellular matrix, in particular of the basement membrane and the adjacent interstitial matrix, are essential prerequisites for a proper function of tissues. Invasive growth in malignant tumors is associated with a destruction of various matrix structures. Due to extensive recent analyses significant advances have been made in the knowledge of the structure of the extracellular matrix, the composition of its most important constituents, their metabolism and that of matrix degrading enzymes. This information provides insight into the pathophysiology of malignant growth. Thereby, it has been shown that malignant tumor growth is associated with a loss of basement membrane (BM) material which, however, disappears not homogeneously, but affects various BM components to different degree. The loss of an intact BM as the first barrier is therefore the initial step of tumor invasion. Despite this loss there is evidence that the de novo synthesis of BM constituents in tumor and adjacent stromal cells is enhanced. Thus, it is obvious that BM material is degraded during the invasion process to significant degree. In addition, since there is a positive correlation between the amount of retained peritumoral BM and a higher degree of tumor cell differentiation the amount of retained BM material seems to represent a marker for the biological behaviour of the tumor cells. The loss of BM material is well explained by a significant expression of major matrix degrading enzymes, the matrix metalloproteinases (MMPs) both on the mRNA and protein level. Here again, there is considerable data indicating that both tumor and stroma cells are involved in the MMP synthesis. In addition to the loss of BM substances, the interstitial extracellular matrix (ECM) is disarranged. This disarrangement may comprise enhanced de novo synthesis ("desmoplasia") or dissolution by distinct MMPs (collagenases, such as MMP-1) reflecting obviously different reaction statuses of the stromal cells. Finally, significant work has been done on the elucidation of the role of regulating cytokine systems. To this regard, particular attention has been paid to the TGF-beta system and it has been shown that the major three isoforms of TGF-betas are upregulated both in tumor and stroma cells. Since the TGF-beta-effect is mainly mediated by a particular signalling system via the TGF-beta-receptors (TBRs), the investigation of this system has provided considerable insight into the role of TBRs which are now known to represent the most potent tumor suppressor genes. Thus frequent mutations in the TBR-II gene, one of the three TBRs, in various carcinomas suggest that these molecular alterations are responsible for both the loss of the control of cellular proliferation (in tumor cells) and altered matrix metabolism (in tumor and stroma cells). The further analysis of this major cytokine system therefore will provide us with major insights into the molecular abnormalities of invasive tumor growth.

Anthropological and palaeopathological analysis of the human remains from three "Tombs of the Nobles" of the necropolis of Thebes-west, upper Egypt.

During several recent excavation campaigns at the necropolis of Sheikh-Abd-el-Gurna, Thebes-West, Upper Egypt, we investigated the human remains of three "Tombs of the Nobles" totalling at least 273 individuals. The investigation covered the human material (skeletons and mummy residues) from the tombs TT-84, TT-85 and TT-95. These tombs had been built in the New Kingdom (approx. 1500-1000 B.C.) and used until the Late period (up to 330 BC). All samples were analyzed macroscopically, isolated findings were further investigated by endoscopic and radiological techniques. The at least 273 individuals covered an age range from newborns to senile individuals with a main age of death between 20 and 40 years of age. The rate of infants and subadults was at 20.2% of all individuals and there was a slight male predominance comprising 54.5% of the adults. In this population a fairly high rate of pathological lesions was seen. Thus, dental conditions generally were poor with a high degree of dental abrasion, an also high rate of carious dental lesions (affecting between 13.8% and 27.7% of the yaws) and consequently a significant number of dental abscesses (mean 15.9%). Residues of trauma were observed in a considerable number of individuals ranging between 12.3% and 22.6% depending on the burial place (mean 15.8%). Inflammatory bone reactions (except the dental abscesses) were present to variable extent, in some locations ranging up to 6.8% of the cases (mean 5.1%). In addition, we noted several cases showing cribra orbitalia (mean 29.2%) and porotic hyperostosis (15.4% of cases), mild to severe osteopenia (7.5%) and in several cases subperiosteal new bone formation suggestive of chronic vitamin D-deficiency ("scurvy") (9.5%). The data support the notion of a significant impairment of living conditions in a high number of individuals. The rates of osteoarthrotic joint alterations were considerably variable depending on the burial places (between 1.9% and 18.5%) providing insight into the physical activities of subpopulations in different locations of the burial places. Similarly, the rate of spondylosis showed also variable extent ranging between 12% and 66.1% of vertebral bodies. In general, our analysis provides evidences that the living conditions in the ancient Egyptian population of one of the main necropoles of Thebes analyzed in this study were quite poor. This is somewhat in contrast to the usually assumed high standard of living of the socially higher population of the capital of ancient Egypt.

Microvessel density and endothelial basement membrane composition in laryngeal squamous cell carcinomas.

In this retrospective study we morphometrically evaluated the density of small blood vessels (microvessel density; MVD) in laryngeal squamous cell carcinomas (SCC) in order to find out if the vascular density provides relevant information on the biology and prognosis of this tumor type. In addition, we investigated the composition of the endothelial basement membrane (BM) to analyze qualitatively the role of the major BM components during tumor-associated angiogenesis. For the immunohistochemical staining of the blood vessels we used the endothelial cell marker CD31 and the BM was analyzed by immunostaining for the major components collagen-IV, laminin-1 and perlecan. Beneath the normal mucosa a mean of 18 vessels/0.41 mm2 was found. Invasive carcinomas showed a significant increase in blood vessel density to 36.3 vessels/0.41 mm2 regardless of the degree of tumor cell differentiation. Interestingly, the highest vascular density was found beneath dysplastic, but non-invasive epithelia (45.5 vessels/0.41 mm2), particularly when associated with a strong lymphoplasmacytic cell infiltration. Statistical analysis revealed a highly significant correlation between MVD and the lymphoplasmacytic infiltrate. However, we did not find any significant correlation between MVD and other clinical/biological parameters. Qualitative analysis of the endothelial BM revealed an "earliest" occurrence of laminin-1 in the obviously newly formed vessels, while collagen IV and perlecan occur at more pronounced stages of vessel formation. There was, however, no prognostically relevant information available from endothelial BM analysis. In summary, at present we do not consider the MVD in SCC of the larynx to be a prognostic factor for the individual outcome of the patient.

Malignant tumors in an ancient Egyptian population.

Since it is still an open debate whether malignant tumors are mainly influenced by environmental factors, the frequency of such malignant tumors in historic populations with different living conditions is of particular interest. In the present study, we investigated the occurrence of malignant tumors affecting bone tissue in a population of mumrnies and skeletons, which had been excavated from the large necropolis of Thebes-West, Upper Egypt. Our study material comprised a series of at least 415 individuals (thereof 325 adults) dating from approx. 1500-500 B.C. All individuals had been mummified, but were severely damaged and partially broken by grave robbers, so that often only parts of the mummies/skeletons were available for investigation. The available specimens were subjected to careful macroscopic examination, while isolated findings were radiologically analyzed. Using this approach, we identified at least 4 cases showing malignant tumors affecting the skeleton. In two cases, multiple mixed osteolytic-osteoblastic lesions suggested multiple metastases from carcinomas. Two further individuals presented with multiple osteolyses (vertebra, pelvis, skull) most suggestive of multiple myeloma. The observation of at least 4 cases of malignant tumors with osseous manifestation in a series of 325 adult individuals provides clear evidence that malignant tumors were not a rare event in the ancient Egyptian study population, particularly when the limitations of a study of tumors manifested only in osseous remnants are taken into consideration. A calculation of the age- and sex-adjusted tumor frequency in our material in comparison with a recent model for such a material by Waldron (1996) indicates that the rate of malignant tumors with bone affection in our series is higher than in an English population from 1901-1905, although lower than in a comparable present day population. This clearly indicates that important factors affecting malignant tumors were effective even in historic populations.

Morphological aspects of altered basement membrane metabolism in invasive carcinomas of the breast and the larynx.

In the present study we compared the localization of major basement membrane (BM) components and their mRNAs between invasive carcinomas of the breast (adenocarcinomas) and larynx carcinomas (squamous cell carcinomas, SCC), in order to determine the extent of BM production and deposition in malignant tumors of biologically different behaviour. Thus, breast carcinomas usually show a rapid locoregional/systemic spread, while the laryngeal SCCs normally show a more locally restricted growth pattern. While normal mammary glands and laryngeal mucosa revealed an intact epithelial BM as evidenced by a continuous linear staining for collagen IV, laminin-1, heparan sulfate proteoglycan (perlecan) and fibronectin-as well as collagen VII in the larynx mucosa-, this continuous staining was lost in the invasive carcinomas, however, affecting the two tumor types differently. In the breast carcinomas, a complete loss was seen even in well differentiated tumors affecting the various BM components similarly, while in the SCCs well differentiated carcinomas had retained significantly more BM material than poorly differentiated ones. In the SCCs, an "early" loss of collagen VII contrasted with a "later" loss of collagen IV, laminin, perlecan and fibronectin the extent of which was, however, associated with a decreasing degree of differentiation. In contrast to the protein findings, by use of the in-situ hybridization we observed a significant expression of mRNA for collagen IV, perlecan and fibronectin. The resulting pattern was comparable between both tumor types and not significantly related to the tumor cell differentiation. Both tumor cells and stroma cells were positively labelled with a more extensive labelling of the stroma cells. Our observations indicate a similar upregulation of the mRNAs for BM-components in breast and larynx carcinomas, but significant differences in the BM-protein deposition so that either major differences in presumed BM-proteolysis or further translational defects are suggested. Furthermore, it can be speculated that the far lesser amount of BM-material in the breast carcinomas may be linked to the more aggressive metastatic spread of those tumors, particularly when compared to the SCCs.

In situ apoptotic and proliferation index in laryngeal squamous cell carcinomas.

The rate of cellular growth is mainly influenced by the balance between cell proliferation and cellular decay. Since to our knowledge, no study so far has analysed the rate of proliferation and apoptosis in the normal laryngeal mucosa and in invasive laryngeal carcinomas, we performed a morphological analysis on both parameters in biopsies from 30 patients with laryngeal carcinoma. We applied the TUNEL end labelling technique for the investigation of apoptosis and immunohistochemistry (Ki-67 antigen) for the determination of the cell proliferation. In our study we demonstrated that invasive tumour growth of the larynx coincides with an increase of both cellular proliferation and apoptosis. Both parameters, however, affected various tumour areas differently. While there was a preferential expression of the Ki-67 antigen at the tumour-stroma interface, apoptotic figures could be found randomly distributed in the tumour. This indicates that the replication of tumour cells and tumour cell decay are differently distributed and possibly independently regulated. Since we observed a particularly strong increase of cell proliferation at the tumour-stroma interface which outnumbered the corresponding rate of apoptosis by far, the enhanced cell proliferation at the tumour border seems to be a main factor for tumour growth. A statistical evaluation revealed significant correlation between the apoptotic index and the degree of tumour cell differentiation, indicating that a high rate of apoptosis coincides with a high level of tumour cell differentiation. There was, however, no statistically significant correlation between prognostic clinical parameters and the rate of apoptosis or that of proliferation.

Prognostic aspects of the loss of epithelial basement membrane components in preinvasive and invasive laryngeal carcinomas.

The integrity of the epithelial basement membrane (BM) is an essential criterion or the biological behaviour of tumors. Previous studies on various types of carcinomas have demonstrated a good correlation between the amount of retained BM and the course of tumor growth. We therefore evaluated the prognostic significance of the tumor BM in laryngeal carcinomas. In this study, we analyzed 66 cases of laryngeal carcinomas using immunohistochemistry for the visualization of the major BM components collagen IV and VII, laminin-1, heparan sulfate proteoglycan (HSPG, perlecan) and fibronectin. The extent of retained BM-material was quantified morphometrically. A subsequent statistical analysis correlated the immunohistochemical findings with clinical and routine histological parameters, such as the mode of tumor infiltration. All carcinomas showed a defective epithelial BM. In addition, we observed a correlation between the degree of tumor cell-differentiation and the amount of BM material retained. The loss of BM, however, affected the various components differently with an "early" loss of collagen VII. In non-infiltrative dysplastic lesions focal BM disruptions were seen which affected the various BM components very similarly. When we statistically analyzed the correlation between the BM staining pattern and prognostically relevant parameters, collagen VII represented a marker for "early" stroma invasion. It also positively correlated with tumor size/stage, presence of lymph node metastasis and the recurrence of tumor growth. The collagen IV expression was positively correlated with the degree of tumor cell differentiation. The other parameters did not show further prognostically relevant data. Our observations provide significant information on the biological course of the disease. Thus, collagen VII may be a marker for "early" invasive tumor growth, as well as for lymphatic metastasis and local tumor recurrence, while the amount of collagen IV correlates with the tumor cell differentiation.

Patients with diabetes-induced microangiopathy show a reduced frequency of carcinomas.

Contradictary results have been reported indicating both increased and reduced risks for malignancies in diabetic patients. This may possibly be due to difficulties in the clinical diagnosis of carcinomas and inaccuracies in the determination of diabetic conditions in the autopsy studies. Since glomerular microangiopathy is a typical feature of long-term diabetes, we performed a retrospective statistical analysis on 5000 consecutive, non-selected autopsy cases with particular reference to the presence/absence of microangiopathy in diabetic individuals. In our study group, we found a total incidence of 9.8% (n = 488) diabetic patients of which 213 (4.3%) had a histologically confirmed significant glomerulosclerosis and a total of 34% patients with verified carcinoma (n = 1699). The age- and sex ratios were matched between diabetic, non-diabetic and carcinoma patients. Systemic and coronary arteriosclerosis were significantly higher in diabetics than non-diabetics (p < 0.0001). Most interestingly, the rate of carcinomas in the diabetic group with nodular and diffuse glomerulosclerosis was 2.5- (p < 0.0001) and 1.9-fold (p < 0.0027), respectively, lower than in the non-diabetic group. In addition, the statistical evaluation showed in the glomerulosclerotic diabetic group significantly lower rates of metastasis. Our retrospective statistical analysis on an unselected series of autopsy cases thus provides evidence that diabetes mellitus with glomerulosclerosis is associated with a significantly lower frequency of carcinomas when compared to individuals without renal microangiopathy. Since TGF-beta is assumed to play a crucial role both in diabetes and carcinogenesis/tumor progression, our findings suggest an altered cell-matrix interaction in diabetes, possibly exerted by chronic TGF-beta overexpression.