Phantoms in the mortuary--DNA transfer during autopsies.

DNA evidence frequently plays an important role in criminal investigations and in some cases may be the only means of convicting a suspect. The constant improvement of DNA analysis techniques affords the individualization of minute amounts of DNA, aggravating the risk of contamination artifacts. In our study, we investigated the prevalence of DNA contamination in the autopsy facilities of the Institutes of Legal Medicine in Essen and Kiel (Germany). Using DNA-free swabs, we took samples from instruments used during autopsy and autopsy tables. Surfaces and instruments were routinely cleaned before sampling. Swabs were subjected to different PCRs to quantify the total amount of DNA and to amplify individual specific STR-markers. In most samples, alleles that could be linked to bodies that had been autopsied before were found. Furthermore, we could show that a DNA transfer from the autopsy table to a body was detectable in four out of six cases investigated. The interpretation of DNA typing results may thus be severely complicated. To avoid DNA contamination, we tried out different cleaning methods, of which only a bleach containing cleaner showed sufficient results.

The role of annexins I, II and IV in tumor development, progression and metastasis of human penile squamous cell carcinomas.

PURPOSE: The outcome of patients with penile squamous cell carcinomas (PSCC) largely depends on occurrence of metastasis. Therefore, prognostic markers indicating the risk for tumor cell spreading would be useful. Since Annexins are potential prognostic markers in a variety of tumors, we immunohistochemically examined the expression of Annexins I, II and IV (ANX AI, ANX AII and ANX AIV) in PSCC. METHODS: Samples originated from 29 patients subjected to surgical resection of invasive PSCC. Immunohistochemistry was done on paraffin-embedded sections using monoclonal antibodies against ANX AI, ANX AII and ANX AIV. Expression of ANXs was compared with clinical data. RESULTS: ANX AI expression was found in conventional PSCC and was absent in basaloid and sarcomatoid subtypes. High ANX AI score was significantly associated with higher T stages (P = 0.006). Strong expression in the invasion front of carcinomas was significantly associated with the occurrence of lymph node metastasis (P = 0.001). ANX AIV expression was weak in conventional PSCC, while it was strong in basaloid and sarcomatoid subtypes. Strong expression of Annnexin IV in the invasion front also showed a significant association with metastasis (P = 0.019). CONCLUSION: Expression of ANXs was different in histologic subtypes of penile carcinomas. Strong expression of ANX AI and ANX AIV in the invasion front seems to indicate a higher risk of lymph node metastasis.

Down-regulation of the metastasis suppressor protein KAI1/CD82 correlates with occurrence of metastasis, prognosis and presence of HPV DNA in human penile squamous cell carcinoma.

In penile squamous cell carcinoma (PSCC), the outcome largely depends on early detection and resection of inguinal lymph node metastases. We investigated the role of metastasis suppressor protein kang ai 1 (KAI1)/cluster of differentiation 82 (CD82), which is known to be of prognostic significance for a wide variety of cancers. Moreover, we analysed the tumours for human papillomavirus (HPV) DNA and loss of heterozygosity at the 11p11.2 locus. Tissue samples of 30 primary PSCCs were investigated immunohistochemically using an anti-KAI1/CD82 polyclonal antibody. The expression was assessed according to the degree of KAI1/CD82-positive tumour cells as positive, decreased or negative. The presence of HPV6/11, HPV16 and HPV18 DNA was analysed by polymerase chain reaction. All patients with decreased or negative expression of KAI1/CD82 in primary lesions had lymph node metastases (p = 0.0002). Patients with positive KAI1/CD82 expression showed a significant better prognosis for survival compared to the other groups (p = 0.0042). Presence of HPV DNA was associated with decreased or negative KAI1/CD82 expression. Lacking or decreased expression of metastasis suppressor gene KAI1/CD82 appears to be a prognostic parameter for the occurrence of lymph node metastases in PSCC. Our study suggests an association of decreased KAI1/CD82 expression with tumour progression, development of metastases and disease-specific death.

Expression of proliferation marker Ki67 correlates to occurrence of metastasis and prognosis, histological subtypes and HPV DNA detection in penile carcinomas.

Clinical outcome of penile squamous cell carcinoma (PSCC) largely depends on the presence of lymph node metastasis. In search of a valuable marker predicting the risk for metastasis, the expression of Ki67 was investigated immunohistochemically in primary tumors and compared to presence of inguinal lymph node metastasis. As human papilloma virus (HPV) is thought to affect Ki67 expression, we evaluated whether occurrence of HPV DNA correlates to Ki67 score or metastatic potential. Samples originated from patients subjected to resection of invasive SCC of penis. Immunohistochemistry was done on paraffin-embedded sections using a monoclonal antibody against Ki67. After DNA isolation from paraffin embedded tissue the presence of HPV 6/11, HPV 16 and HPV 18 DNA was analyzed by PCR. Statistical analysis was done using two tail unpaired t test and Chi-square test. Four of 28 patients showed a weak Ki67 expression, without displaying lymph node metastasis. Among 17 patients showing an intermediate Ki67 index, eight exhibited metastases while in all seven patients with a strong expression of Ki67 lymph node metastases were found. The median Ki67 expression in metastastic lesions was significantly different (50.3%) from tumors without lymph node metastasis (31.8%) (p=0.024). Furthermore, a correlation between presence of HPV DNA and strong Ki67 expression was determined (p=0.009). Since our study demonstrated a strong Ki67 labeling index significantly associated to positive lymph nodes, we suggest Ki67 expression as a prognostic marker for lymph node metastasis in penile squamous carcinoma.

Expression of Skp2 and p27KIP1 in naevi and malignant melanoma of the skin and its relation to clinical outcome.

Skp2 (S-phase kinase associated protein 2) controls progression from G- to S-phase by promoting the proteolysis of the cyclin dependent kinase inhibitor p27KIP1. Despite the fact that a p27KIP1 decrease has been documented in melanoma progression, the role of Skp2 in these tumours is unknown. We therefore examined by immunohistochemistry the expression of Skp2, p27KIP1 and Ki-67 in 10 naevi (Ns), 15 superficial spreading melanomas (SSMs), 10 nodular melanomas (NMs) and 14 melanoma metastases (Ms). Nuclear Skp2 expression augmented with increasing malignancy (Ns: 1.4%, SSMs: 5.6%, NMs: 17.3%, Ms: 19.1%). In all tumours nuclear Skp2 expression correlated with Ki-67 (p=0.024) and inversely with p27KIP1 (p=0.007). A cytoplasmic reaction for Skp2 was also observed in most tumours and its expression decreased from Ns (12.3%) to SSMs (7.9%) and NMs (4.5%). In contrast, Ms showed an increase of cytoplasmic Skp2 (11.9%) that correlated with its nuclear expression (p=0.016). While nuclear Skp2 expression correlated with the pT-level (p=0.023), Clark-level (p=0.023) and Breslow index (p=0.019), the cytoplasmic Skp2 expression might be of biological significance only in NMs since it correlated with tumour depth (p=0.02) and pT-level (p=0.025). Our data suggests that Skp2 could contribute to melanoma progression. This is further highlighted by the fact that vertical growth phase (VGP) melanomas show significant higher nuclear Skp2 expressions when compared with the harmless radial growth phase (RGP) (p=0.047). Also nuclear Skp2 expression correlates with a reduced survival time (p=0.025) in melanoma.

Can different genetic changes characterize histogenetic subtypes and biologic behavior in sporadic malignant melanoma of the skin?

In sporadic malignant melanoma, different chromosomal regions with nonrandom aberrations have been discovered, including 1p36, 6q, 9p and 10q. First results provide a genetic basis for the concept of primarily vertical, biologically aggressive melanomas and radial growing, mostly benign melanomas. These are mainly represented by nodular melanoma (NM) and early superficial spreading melanoma (SSM), respectively. Deletions in 1p36 could be found only in NMs and metastatic melanoma. Aberrations of chromosome 10 occur predominantly in NMs, whereas deletions on chromosome 9 are more frequent in SSMs. Despite a variety of genes tested, neither a tumor suppressor gene with importance in all malignant melanomas of the skin nor one clearly defining the transition from the radial growth phase to the vertical growth phase has been determined. Nevertheless, the pattern of genetic alterations may soon lead to finding such genes and development of drugs targeting these genes or their products, which would be of great benefit to melanoma patients.

Lhermitte-Duclos disease in 3 children: a clinical long-term observation.

We report three boys in whom a diagnosis of Lhermitte-Duclos disease (LDD) was assumed from characteristic neuroimaging findings. LDD was confirmed by an open biopsy in patient 1, while a biopsy in patient 2 was inconclusive. Histologic confirmation in patient 3 was deliberately not attempted. However, a follow-up observation of stable clinical and neuroimaging findings over 2, 5 and 11 years, respectively, support the diagnosis of LDD. Despite extensive expansion of the lesion with brainstem involvement, clinical signs in two boys were minimal, while one patient has cognitive impairment and a complex oculomotor disturbance. So far we found no evidence for an association with Cowden disease (CD). No germline PTEN mutations were detected in these children, but the amount of available biopsy tissue in patients 1 and 2 was insufficient for a complete genetic analysis of tumor tissue. In conclusion, LDD can usually be diagnosed by MRI. In view of the favourable natural history, a conservative "wait and see" strategy is justified, particularly if radical tumor resection is not possible. LDD is often not associated with CD and germline PTEN mutations seem not to be present in isolated LDD.

Clear cell chondrosarcoma of the larynx: a case report of a rare histologic variant in an uncommon localization.

The authors describe a clear cell chondrosarcoma of the larynx. The clear cell type is a rare variant of chondrosarcoma that only twice has been reported in this localization. The light-microscopic diagnosis of the actual case was confirmed by immunohistochemical results, in particular by positive staining for S-100 protein and collagen type II, and ultrastructural findings. Loss of heterozygosity analysis demonstrated allelic loss at 9p22 and 18q21, but neither in the region of the Rb gene on chromosome 13q nor at the p53 locus on chromosome 17p where allelic loss has already been reported in chondrosarcomas. Furthermore, our molecular genetic investigations revealed a methylation of the cell cycle control gene p16, which is localized on chromosome 9p. This characteristic has been recorded previously only in high-grade chondrosarcomas. Mutations in the exons of p16, alterations of the putative tumor suppressor gene MMAC1/PTEN on chromosome 10q, or an amplification of the cyclin D1 gene (CCND1) on 11q13, which were found to be changed in other studies of chondrosarcomas, could not be demonstrated here.

Multiple chromosomal underrepresentations detected by interphase cytogenetics - possible prognostic markers in head and neck tumors?

Relevant prognostic factors for head and neck squamous cell carcinoma are tumor extension (pT), occurrence of lymph node metastases (pN) and grade of differentiation (G). We tried to correlate these histological characteristics with numerical aberrations of whole chromosomes as demonstrated by fluorescence in situ hybridization techniques (FISH). Therefore, we investigated isolated interphase cells from paraffin sections of squamous cell carcinomas of the head and neck region from 46 patients with centromeric DNA probes for chromosomes 1, 3, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X and Y. The majority of tumor samples showed aneuploidy for most chromosomes analyzed. The main chromosomal abnormality was loss of chromosomal material, predominantly of chromosomes 3 (28%), 6 (20%), 9 (26%), 10 (24%) and 18 (33%). Multiple deletions could be demonstrated more frequently in poorly differentiated carcinomas (88% G3-tumors with more than one deletion in contrast to 66% G2-tumors). The occurrence of multiple deletions may also correlate with progression in lymph node metastasis (66% in pN0-tumors vs. 85% in pN2-tumors), whereas the differences between the stages of primary tumor extension were not so obvious. Despite of a some-what disproportionate distribution of tumors in the different pT- and pN-stages and the rather low number of cases, our results suggest a relationship between the quantity of chromosomal underrepresentation, grade of differentiation and higher lymph node stage. Therefore, they underline the importance of chromosomal deletions as a possible additional prognostic marker in head and neck squamous cell carcinoma.

PTEN/MMAC1 in malignant melanoma and its importance for tumor progression.

A novel tumor suppressor gene, PTEN/MMAC1, on 10q23, displayed a number of mutations in solid tumors as gliomas and breast cancer. Aberrations of the long arm of chromosome 10 have been frequently detected in tumor progression of malignant melanoma of the skin by a variety of methods including cytogenetic analysis, fluorescence in situ hybridization and loss of heterozygosity analysis. Compared to previous studies, which propose an involvement of PTEN/MMAC1 in malignant melanoma mostly on the basis of data derived from cell lines and metastases, we analyzed a broader spectrum of exclusively patient derived tumor tissue by PCR and direct sequencing analysis of PTEN/MMAC1. Here, we present data of 25 primary melanomas (8 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases of 41 patients. Neither loss of the complete gene nor a whole exon nor any nonsense mutations could be demonstrated. However, we detected several polymorphisms and some mutations in the introns, and in two metastatic tumors mutations with an amino acid change. Our results obtained from tissue samples underline that mutations of PTEN/MMAC1 are not an essential event in the onset of malignant melanoma of the skin, but could have an impact on tumor progression.

Analysis of microsatellite polymorphism in red deer, roe deer, and fallow deer -- possible employment in forensic applications.

DNA microsatellites play a major role in population genetics, linkage mapping, and parentage studies of mammals. In addition, they may be used for forensic purposes, if an individual identification of a specific animal is necessary. Therefore, we tested a variety of microsatellite polymorphism derived from reindeer (Rangifer tarandus) by PCR and sequencing analysis for use in red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama). Twelve of these microsatellites were selected for further analysis. In all these microsatellite polymorphism short tandem repeats could be detected for one or all three species as shown by sequencing analysis. In red deer, more than two alleles were found in eight microsatellites, in roe deer more than two alleles could be demonstrated in seven microsatellites, whereas in fallow deer more than two alleles were found in only two microsatellite polymorphism. A comparison of sequences of PCR products from the three deer species with the sequences of reindeer revealed several differences between the four species. In six microsatellites -- selected because or their reliability in PCR and because of their polymorphic character -- we established a sequenced allelic ladder and give population data of all three species from 82 deer of the Northeast region of Germany (Vorpommern). Our results show the possibility to use microsatellite polymorphism in the identification of deer in forensic applications like poaching.

TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin.

A novel candidate tumor suppressor gene, TTC4, on chromosome 1p31 has been described recently. Since aberrations in this region have been detected in malignant melanoma, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 32 primary melanomas (15 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis for mutations in all exons of TTC4. Tumors comprised a wide range of thickness (Breslow index) and Clark levels. No mutations could be detected in typical or atypical naevi, but we found seven different point mutations in the tumor samples, six of them causing an amino acid change. Ten melanoma samples belonging to nine patients showed one or more of these mutations. In detail, in six of 25 metastases, in two of 17 nodular melanomas and in two of 15 superficial spreading melanomas point mutations could be detected. In two cell lines, a loss of a whole exon could be demonstrated and in one cell line we found a point mutation. In addition, three polymorphisms were found. Our findings indicate that TTC4 may participate in the pathogenesis of malignant melanomas of the skin.

[Individual identification of fallow deer using microsatellite polymorphism in a case of poaching]]. / Individualisierung von Damwild mit Hilfe von Mikrosatellitenpolymorphismen in einem Fall von Wilderei.

The paper reports on molecular biological investigations in a case of poaching which resulted in considerable damage for property. Blood traces from poaching sites have been analysed and compared with blood from two knives and a pair of trousers of the suspects. In two of three poaching sites, genomic DNA could be amplified by PCR and assigned to fallow deer. The authors could demonstrate identical allelic combinations between one of the poaching sites and a part of the traces. Problems of the legal appreciation in this case are described.

First hints for a correlation between amplification of the Int-2 gene and infection with human papillomavirus in head and neck squamous cell carcinomas.

Infection with human papillomavirus (HPV) and alterations in certain genes have frequently been proposed as mechanisms in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Here, we investigated 47 HNSCC for the presence of HPV and, by fluorescence in situ hybridisation, for amplification of Int-2 and Hst-1 in the search for a possible correlation. The highest frequency of HPV infection was found in hypopharyngeal carcinomas, while amplification of Int-2 or Hst-1 was distributed more equally among the different localisations. Amplification of Int-2 was detectable (7 of 9 cases) in 78% of the HPV-positive carcinomas, whereas no virus infection could be found in the five cases with amplified Hst-1 only. In spite of the rather low number of infected tumour samples, our results suggest a correlation between HPV infection and amplification of Int-2.

Use of interphase cytogenetics in demonstrating specific chromosomal aberrations in solid tumors--new insights in the pathogenesis of malignant melanoma and head and neck squamous cell carcinoma.

The detection of structural and numerical chromosomal aberrations is an important part of the characterization of tumors and genetic diseases. The direct demonstration of DNA sequences in interphase nuclei and metaphases by fluorescence in situ hybridization (FISH) has been termed interphase cytogenetics. It has been proven as a powerful technique to detect specific aberrations in a wide variety of cell types, including paraffin-embedded tissue. Nowadays a standard method in leukemia and lymphoma, interphase cytogenetics contributes mainly to the diagnosis in these tumors and helps to classify soft tissue tumors. Therefore FISH is mandatory for the choice of therapy in these tumors. In contrast to the aforementioned, up to now, the value of FISH in solid tumors is mostly limited to pure research and contributes in this way to our understanding of tumor biology. But with the use of paraffin-embedded tissue and the first results obtained, it seems very likely that a direct correlation between histological classification and cytogenetic characteristics of solid tumors can be achieved in the near future. This information might not only provide insights into tumor biology, but could also contribute to a different tumor classification, a sort of risk estimation, where we might predict the possible biological behavior of solid tumors. This could greatly influence further therapeutic decisions thus establishing the FISH technique as an indisputable part in the diagnosis of solid tumors.

Mitochondrial DNA in the Central European population. Human identification with the help of the forensic mt-DNA D-loop-base database.

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.

Significance of the small subtelomeric area of chromosome 1 (1p36.3) in the progression of malignant melanoma: FISH deletion screening with YAC DNA probes.

The short arm of chromosome 1 (1p), especially the subtelomeric region of 1p36, is a common site for abnormalities in malignant melanoma of the skin. In a recent study nodular melanomas displayed deletions of 1p36 in an augmented percentage of cases. To evaluate the dimension of these deletions and to study their significance for the progression of malignant melanoma we analyzed seven melanoma cell lines, 32 primary tumors, and 32 metastatic tumors by fluorescence in situ hybridization with the DNA probe D1Z2 in 1p36.3 and eight YAC DNA probes hybridizing to 1p36, 1p32, 1p31, and 1p21. All cell lines, 91% of the metastatic tumors and 63% of nodular melanomas showed a deletion of 1p36.3. In the YAC hybridization experiments, the most frequent deletions were found in 1p36 in all cell lines, in 13% of nodular melanoma, and in 44% of metastatic tumors. Deletions in 1p36 were mostly confined to a rather small area near the locus D1Z2. The frequent occurrence of this deletion in melanomas with a high metastatic potential and the abundant accumulation of this deletion in metastasis point to genes located on 1p36, which might be of significance for the metastatic capability of malignant melanoma.