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.

Non-diagnosed pulmonary hyalinizing granuloma (PHG) as a cause of sudden unexpected death.

Pulmonary hyalinizing granuloma (PHG), a very rare benign tumour of the lungs, was first reported in 1977. We present a PHG of a 32-year-old woman from Yemen who collapsed 1 day after her arrival in Germany. Tuberculosis was suspected and the health authorities nearly closed part of one of the major international airports in Europe. However, this drastic measure was avoided by autopsy and a correct interpretation of the solid-elastic and well-circumscribed lung tumour as not characteristic for tuberculosis. Although the final diagnosis of PHG was only achieved after histology, this case strongly illustrates the necessity of a profound morphological training of forensic physicians.

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 annexin AI in conventional renal cell carcinoma (CRCC) correlates with tumour stage, Fuhrman grade, amount of eosinophilic cells and clinical outcome.

There is increasing evidence that Annexin AI (ANX AI) expression is dysregulated in several carcinomas and tumour cell lines. In order to gain insight into the putative role of ANX AI in tumorigenesis, clinical outcome and metastatic potential of conventional renal cell carcinomas (CRCCs) we investigated the expression of ANX AI in CRCCs and metastases. Furthermore, it was elucidated whether ANX AI overexpression affects migratory potential in Caki-1 cells. ANX AI immunohistochemistry was performed on 33 samples of CRCCs and 10 metastases. ANX AI expression was assessed in 12 samples by 2-dimensional gelelectrophoresis (2-DE), subsequent mass spectrometry and RT-PCR. Immunohistochemical data were statistically correlated with pathological parameters, amount of eosinophilic cells and clinical outcome. Furthermore, a haptotactic migration assay was done on Caki-1 cells transfected with ANX AI. Immunostaining for ANX AI was found in 18 tumours and all metastases investigated. Intensity of immunohistochemical staining correlated to Fuhrman grade, amount of eosinophilic cells and clinical outcome. 2-DE and RT-PCR confirmed the presence of ANX AI in neoplastic tissue. Overexpression of ANX AI did not significantly influence cell migration. From these findings ANX AI expression seems to be related to Fuhrman grade, clinical outcome and metastatic potential of CRCCs. Thus ANX AI could serve as a prognostic marker for tumour progression.

Non-diagnosed pheochromocytoma as a cause of sudden death in a 49-year-old man: a case report with medico-legal implications.

Pheochromocytomas are known to be rare causes of sudden death. A 49-year-old man with a medical history of arterial hypertension and diabetes mellitus complained about nausea and malaise in the morning. During the day his condition deteriorated. He went to the emergency department, where he was given intravenous drugs against nausea and was sent home. On the way back, his condition deteriorated dramatically so that his wife drove back to the emergency room, where he collapsed and sustained cardiac arrest; resuscitation efforts were unsuccessful. Autopsy revealed a large tumour of the left adrenal gland. The strong suspicion of pheochromocytoma was confirmed by histology, immunohistochemistry and biochemical investigations. An acute hypertensive crisis, caused by the hitherto unknown pheochromocytoma was ascertained as the cause of death. The morphological findings are presented, the difficulty to diagnose pheochromocytoma and the medico-legal implications are discussed.

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients.

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.

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.

Expression of annexin II in conventional renal cell carcinoma is correlated with Fuhrman grade and clinical outcome.

Conventional renal cell carcinomas (CRCCs) were investigated for the expression of annexin II (ANX II) to determine out whether this calcium-binding protein could serve as a useful prognostic marker. CRCCs and adjacent nonneoplastic tissue from 33 patients were investigated for ANX II by immunohistochemistry, RT-PCR, and western blot analysis. ANX II expression was correlated with tumor differentiation (Fuhrman grade) and to clinical outcome. Tumors were composed of ANX II positive and negative cells. In grade I tumors only a weak membranous staining was seen in immunopositive cells. In grade II and III tumors, however, ANX II was seen in the cytoplasm and at the cell membranes of tumor cells. On serial sections membranous and cytoplasmic immunoreactivity for ANX II occurred predominantly in eosinophilic cells whereas clear cells were mostly immunonegative. The ANX II expression in CRCCs was correlated with clinical outcome and Fuhrman grade. Since ANX II expression is correlated with Fuhrman grade and clinical outcome it may be a useful marker for prognosis in CRCC.

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.

[Leiomyoma of the kidney. Differential diagnostic aspects of renal cell carcinoma with increasing clinical relevance]. / Das Leiomyom der Niere. Ein differentialdiagnostischer Aspekt des Nierenzellkarzinoms mit zunehmender klinischer Relevanz.

Renal leiomyoma is a rare benign tumor which has its origin in smooth muscle cells of different structures of the kidney. The clinical incidence of renal leiomyoma is much lower than the frequency described in large autopsy studies. Renal leiomyomas are mainly located in the renal capsula and pelvis or next to those structures. Clinical symptoms are nonspecific (e.g., flank pain or flank tumor) or completely absent, making most of the leiomyomas an incidental finding during routine diagnostics. A differential diagnosis between renal leiomyoma and renal cell carcinoma on the basis of medical imaging is extremely difficult. Therefore, laparotomy and nephrectomy are performed in most cases of suspected renal cell carcinoma. The definitive diagnosis of a leiomyoma is only possible after histological examination of the tumor. Due to the rising number of diagnoses resulting from improved medical imaging, renal leiomyomas are gaining more importance in the differential diagnosis of renal cell carcinoma, especially with respect to kidney-sparing surgery.

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.

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.

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.

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.

Differences in chromosomal aberrations between nodular and superficial spreading malignant melanoma detected by interphase cytogenetics.

At present, little information is available on specific chromosomal aberrations in malignant melanomas of different subtypes and different growth behaviors. Therefore, we have applied fluorescence in situ hybridization on isolated interphase cells from paraffin sections of 79 primary tumors of malignant melanomas: 47 nodular melanomas and 32 superficial spreading melanomas in various stages. We used centromeric probes for the chromosomes 1, 4, 6, 7, 9, 10, 11, 12, 15, 17, 18, X, and Y and a midisatellite probe localized in 1p36. The number of chromosomal aberrations and the ploidy of the cells rose with the tumor stage in both subtypes, although in superficial spreading melanomas, fewer chromosomal abnormalities were detectable than in nodular melanomas. A deletion in 1p36 could only be found in nodular melanomas (mostly in higher tumor stages), not in superficial spreading melanomas. Our results show that the different histologic subtypes of malignant melanoma of the skin differ also in their chromosomal aberrations. In addition, it seems that there may be a correlation between the growth characteristics and putative tumor suppressor genes on 1p36.

An increased frequency of numerical chromosomal abnormalities and 1p36 deletions in isolated cells from paraffin sections of malignant melanomas by means of interphase cytogenetics.

At present, little information is available on tumor and stage-specific chromosomal aberrations in malignant melanoma. Therefore, we applied fluorescence in situ hybridization on isolated interphase cells from paraffin sections of 25 cases of malignant melanomas, comprising 17 primary tumors (PTs) and 8 metastases (MTs) in various anatomical sites. We used centromeric probes for chromosomes 1, 7, 9, 10, 11, 12, 15, 17, 18, X, and Y and a midisatellite probe localized in 1p36. Four of the PTs and 5 of the MTs showed polyploidy for all applied probes. The most frequent type of numerical aberration was an overrepresentation of chromosomes 1 (3 PTs, 5 MTs) and 7 (3 PTs, 1 MT), and an underrepresentation of chromosomes 9 (3 PTs) and 10 (6 PTs, 5 MTs). The Y chromosome was lost in all male tumors. In addition, we observed monosomy 11, 12, 15, 17 or 18, and trisomy 12 or 17. Only 1 PT showed no aberrations for any applied DNA probe. A deletion in the near-telomeric region of 1p36 was found surprisingly often (9 PTs, 7 MTs). Our results suggest that the loss of gene(s) in this region is an important event in the pathogenesis of malignant melanoma of the skin.