Gene-expression signature of adhesion/growth-regulatory tissue lectins (galectins) in transitional cell cancer and its prognostic relevance.

AIMS: Lectins, and especially galectins, appear to be important in malignancy-associated processes. The aim was to analyse comprehensively the presence of galectins in urothelial tumours. METHODS AND RESULTS: Non-cross-reactive antibodies against seven family members from the three subgroups (prototype: galectin-1, -2 and -7; chimera type: galectin-3; tandem-repeat type: galectin-4, -8 and -9) were used. Gene expression was monitored in specimens of normal urothelium, fresh tumour tissue and cell lines by real-time polymerase chain reaction (PCR). The presence and evidence of tumour-associated up-regulation were shown for galectin-1 and -3. This was less clear-cut for galectin-4 and -8. Galectin-7 was expressed in all cell lines; galectin-2 and -9 were detected at comparatively low levels. Galectin-2, -3 and -8 up-regulation was observed in superficial tumours, but not in muscle-invasive tumours (P < 0.05). Immunoreactivity correlated with tumour grading for galectin-1, -2 and -8, and disease-dependent mortality correlated with galectin-2 and -8 expression. Binding sites were visualized using labelled galectins. CONCLUSIONS: The results demonstrate a complex expression pattern of the galectin network in urothelial carcinomas. Galectin-1, -2, -3 and -8 are both potential disease markers and also possible targets for bladder cancer therapy.

[Polyneuropathy as a sole syndrome in malignant thymoma]. / Polyneuropathie als einziges paraneoplastisches Syndrom bei malignem Thymom.

Up to 40% of patients with malignant thymoma suffer from paraneoplastic symptoms (90% myasthenia, 10% other symptoms). A 55-year-old patient developed ascending symmetrical sensorimotor tetraparesis. A malignant thymoma without metastases was diagnosed 6 months later. Despite thymectomy followed by radiation and high-dose corticosteroid therapy, the polyneuropathy progressed. Six months after onset, the patient was bound to a wheelchair. Immunosuppressive therapy with cyclophosphamide was initiated, leading to marked remission. After ten cycles, the patient was able to walk independently with walking aids. After the sixth and tenth cycle, respectively, attempts to discontinue immunosuppression led to relapse. In several diagnostic workups, however, there was no tumour relapse. After 13 cycles, cyclophosphamide was replaced by immunoglobulins (0.4 g/kg per day i.v. for 5 days/month) due to progressive renal failure. The patient died just before the second course of this treatment. In conclusion, in the differential diagnosis of rapidly progressive polyneuropathy, a malignant thymoma should be considered, even in the absence of myasthenia. Immunosuppression with cyclophosphamide resulted in amelioration of symptoms in this patient.

GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis.

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.

Ultrasound imaging of Nd:YAG laser-induced tissue coagulation in porcine livers.

AIMS: Absorption of laser light energy induces denaturation of proteins and thermocoagulation of irradiated tissue. Recently, MRI-guided laser coagulation in combination with MR thermometry was reported as a treatment of liver tumours. In the present study ultrasonographic imaging was evaluated for its suitability in laser induced tissue thermocoagulation. METHODS: Fresh porcine livers were used for ex vivo examinations. Placement of the laser catheter and tissue coagulation during laser light emission were online monitored by ultrasonography. Nd:YAG laser-induced tissue damage was evaluated by macroscopical and microscopical examinations of histological sections. RESULTS: During laser light emission a marked hyperdense signal enhancement was observed by ultrasonography which strongly correlated with the extent of macroscopic tissue damage. The size of laser-induced coagulation zone depended on both the power setting and total energy delivered. Carbonization of the tissue surrounding the laser tip is a limiting factor because of laser light absorption. However our data indicate that using appropriate laser energy and exposure time prevent carbonization although carbonization can not be visualized by ultrasonography. CONCLUSIONS: It is concluded from the present ex vivo studies that laser coagulation can be effectively performed under ultrasonographic guidance.

Relationship between DNA ploidy-related parameters and the deletions in mismatch repair genes MLH1 and MSH2 in lentigo maligna and malignant melanomas.

Defects in DNA mismatch repair genes MLH1 and MSH2, first described in hereditary nonpolyposis colon cancer (HNPCC), have been postulated to be responsible for malignant transformation in several tumours. To date there are no data on cutaneous tumours. Using a PCR assay it was possible to identify deletions in MSH2 (exonic regions 12 and 13) in 16 of 47 lentigos maligna and in 10 of 36 malignant melanomas. Deletions in MLH1 (exonic regions 15 and 16) were found in 11 of 47 lentigos and in 15 of 36 melanomas. Comparison of DNA ploidy-related parameters between lentigos with and without exonic deletions in MSH2 and MLH1 did not show any significant differences. In contrast, melanomas positive and negative for exons 12 and 13 (MSH2) (26/36 and 10/36, respectively) differed significantly with respect to the percentages of diploid cells (P = 0.0179) and tetraploid cells (P = 0.0042). Comparison of melanomas positive and negative for exons 15 and 16 (MLH1) (21/36 and 15/36, respectively) showed significant differences in the percentage of aneuploid cells between 2c and 4c (P = 0.0141) and tetraploid cells (P = 0.0404). In summary, deletions in DNA mismatch repair proteins MSH2 and MLH1 were present both in lentigo maligna and in melanomas and correlated with DNA ploidy-related parameters in malignant melanomas.

Clear-cell odontogenic carcinoma with pulmonary metastases resembling pulmonary meningothelial-like nodules.

Clear-cell odontogenic carcinoma (CCOC) is a rare neoplasm with malignant potential and unknown cytogenetic alterations. We describe the case of a 43-year-old woman who presented with an unusual odontogenic epithelial tumor. Histologically, the tumor was composed of clear-cell areas and exhibited a squamous pattern with little nuclear pleomorphism similar to benign squamous odontogenic tumor. Multiple small pulmonary nodules occurring 3 years after primary surgical treatment histologically closely resembled benign minute pulmonary meningothelial-like nodules (MPMN) with clear-cell features. Comparative genomic hybridization (CGH) and immunohistochemistry, performed as diagnostic adjuncts, revealed in the odontogenic tumor and the pulmonary lesions a very similar pattern of chromosomal aberrations (loss of 9, gains of 14q, 19 and 20 in both, and additional loss of 6 in the odontogenic tumor) and the same pattern of expression (positive for cytokeratin 5, 6, 8, 19 and negative for cytokeratin 18, epithelial membrane antigen, and vimentin), differing from that of MPMN. These findings confirmed the final diagnosis of metastasizing CCOC with partial squamous differentiation, substantiated the unfavorable prognosis of the clear-cell component, and highlighted the diagnostic impact of CGH and immunohistochemistry for classification of these morphologically peculiar pulmonary CCOC metastases.

Relation between DNA ploidy status and the expression of the DNA-mismatch repair genes MLH1 and MSH2 in cytological specimens of melanoma lymph node and liver metastases.

DNA-mismatch repair is essential for preventing genetic instability, and its important protective role has been demonstrated in several tumors. The main aim of this study was to investigate the expression of MLH1 and MSH2 (on the RNA level) in melanoma liver and lymph node metastases, and to define the relation between DNA ploidy status and mismatch repair gene expression. MLH1 was found in 29/33 melanoma lymph node and in 5/17 melanoma liver metastases. MSH2 was present in 26/33 lymph node and 5/17 liver metastases. A comparison of MLH1 and MSH2 positive and negative melanoma metastases showed that there were highly significant differences in the percentages of diploid cells, aneuploid cells between 4c and 8c, octaploid cells, and 5c exceeding rate. This fact confirms the strong relation between the loss of DNA-mismatch repair gene expression and advanced DNA aneuploidy status in melanoma metastases.

Downregulation of the retinoblastoma gene expression in the progression of malignant melanoma.

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.

Tryptophan hydroxylase and 5-hydroxytryptamine in carcinoids of the gastrointestinal tract.

BACKGROUND: Tryptophan hydroxylase is the enzyme that catalyzes the formation of 5-hydroxy-L-tryptophan which is the initial and rate-limiting enzyme in the biosynthesis of 5-hydroxytryptamine (serotonin). The aim of this study was to analyze the differential expression of tryptophan hydroxylase and 5-hydroxytryptamine in human gastrointestinal carcinoids. MATERIAL AND METHODS: Primary carcinoids and hepatic carcinoid metastases (n = 8) were investigated for the presence of tryptophan hydroxylase, 5-hydroxytryptamine and chromogranin A. RESULTS: Only one of the tumors examined was immunoreactive for tryptophan hydroxylase, as determined by immunohistochemical techniques and Western blot analysis. All carcinoids expressed 5-hydroxytryptamine and chromogranin A. CONCLUSIONS: Carcinoids appear to be a heterogeneous group with respect to the expression of this monooxygenase. Based on these results it is proposed that other cells of the intestine are involved in the biosynthesis of 5-hydroxytryptamine precursors taken up by carcinoid tumor cells.

Analysis of the DNA content in the progression of recurrent and metastatic melanomas.

Ploidy status and ploidy related parameters of 18 primary melanomas, 32 recurrences and 18 lymphatic metastases were investigated applying CAS200 image analyzer. All the tumours investigated were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). Primary melanomas differed from recurrent tumours concerning the percentage of aneuploid cells between 4c and 8c and 5c ER. Comparison of cutaneous tumours with lymphatic metastases showed a significant difference concerning the percentage of aneuploid cells between 2c and 4c. An already high aneuploidy rate in primary tumours suggests that recurrent and metastatic clones of cells are present in early stages and that aneuploidy status in primary melanomas could be regarded as one of the risk factors of recurrences and metastases.

Prognostic significance of newly defined ploidy related parameters in melanoma.

5c exceeding rate is the parameter, most frequently showing prognostic impact. The CAS200 image analyzer makes possible the measurement of additional parameters defining single subfractions of cells, as for example the ratios of diploid, aneuploid, tetraploid, octaploid and 16-ploid cells. The main objective of this study was to define the prognostic significance of these new parameters in 106 primary melanomas with known survival time. 29 out of 106 melanomas were euploid. 77 out of 106 showed an aneuploid histogram. Multivariate analysis with Cox regression demonstrated that the percentage of aneuploid cells between 2c and 4c and the percentage of aneuploid cells between 4c and 8c, but not 5c exceeding rate, were able to influence survival time.

Relationship between GAGE-1/-2 expression, EBV infection and interferon-gamma expression in undifferentiated carcinoma of nasopharyngeal type.

GAGE-1/-2 proteins are novel tumour markers, functionally related to tumour rejection. The objective of the present study was to identify the existence of a relationship between GAGE-1/-2 expression, Epstein Barr Virus (EBV) infection and viral infection-induced cytokine expression in cultivated tumour cells and archival specimens of undifferentiated carcinoma of nasopharyngeal type (UCNT). PCR and in situ hybridization techniques were employed. In cultivated UCNT cells, interferon-gamma (IFN-gamma) induced synthesis of GAGE-1/-2 mRNA. In archival tumour specimens (n = 10) however, GAGE-1/-2 gene expression was detected in only 3/8 cases with coincident EBV infection and IFN-gamma expression. In conclusion, EBV infection appears to induce IFN-gamma gene expression in most tumors, but GAGE-1/-2 expression in only some tumours. The role of IFN-gamma and other factors in triggering GAGE-1/-2 gene activation must be elucidated further. The relevance of GAGE-1/-2 gene expression and its detection by PCR for future immunotherapy is discussed.

Proliferative activity in the progression of pigmented skin lesions, diagnostic and prognostic significance.

Proliferative compartments of a tumour can be determined cytophotometrically, by in situ hybridisation or by immunohistochemical detection of Ki67 antigen. The main objective of this study was to analyse the proliferative activity during the progression of pigmented skin lesions with respect to differential diagnostic and prognostic applications. The material investigated consisted of 209 pigmented skin lesions (31 naevi, 30 dysplastic naevi, 106 primary melanomas, 20 lymphatic and 22 organ melanoma metastases). Comparison of the ratios of cells in the S-phase gained by two different methods (cytometry, in situ hybridisation) did not show any significant differences. The correlations between Ki67 and S-phase indices in every diagnostic group were highly significant. The results of forward and backward Cox regression were identical and only Ki67 showed an independent prognostic influence (p < 0.001, coefficient in regression 0.02) with change in risk 2% and confidence limit ranging between 1.1% and 2.9%.

Papillary squamous cell carcinoma of the uterine cervix: report of three cases and a review of its classification.

Papillary squamous cell carcinoma (SCC) of the uterine cervix has been defined as a malignant squamous cell lesion characterized by a papillary architecture with fibrovascular cores and moderate to severe dysplasia devoid of frank keratinization and koilocytic change. Papillary SCC should be histopathologically delineated from other rare variants of SCC with papillary features including verrucous and condylomatous carcinoma and the recently recognized (squamo-)transitional cell carcinoma of the uterine cervix. We report three cases of papillary SCC (FIGO stages IB, IV, and IVB) in postmenopausal women. Each tumor tested was positive for human papillomavirus (HPV) 16 and negative for HPV 6, 11 and 18 by general primer mediated polymerase chain reaction and subsequent enzyme-linked immunosorbent assay (PCR-ELISA). These findings 1) support the hypothesis that papillary SCCs (unlike verrucous carcinoma) are similar with regard to risk factors to (squamo-)transitional and condylomatous carcinoma; 2) suggest that HPV may play an etiologic role in at least some of these tumors; and 3) suggest that papillary SCC is the only subtype among squamous/(squamo-)transitional carcinomas that is associated with high-risk HPV infection in the absence of HPV-related histopathologic alterations.

Differential binding activities of lectins and neoglycoproteins in human testicular tumors.

Testicular germ-cell tumors, a morphologically and clinically diverse group of malignancies provide an ideal model for investigating the biology of glycoconjugates because the biosynthesis of oligosaccharide chains of glycoproteins monitored by plant/invertebrate lectins often changes during tumorigenesis, tumor progression, and metastasis. To investigate such changes in germ-cell tumors, we analyzed 67 surgical specimens from 31 seminomas, 32 embryonic carcinomas, and four choriocarcinomas using glyco- and immunohistochemistry that involved five plant/invertebrate lectins, 16 neoglycoproteins, and galectin-1 antibody. The results showed that some of these markers, such as melibiose-, lactose-, and beta-N-acetylgalactosamine-BSA-biotin were clearly differentially expressed amongst these tumors and between primary and metastatic embryonic carcinomas. The differences in staining for positivity, intensity, and heterogeneity indicate that the differential display of glycoconjugates in tumor cells may be important in tumor growth, metastasis, or prognosis because subtypes of these tumors behave quite differently from one another. Furthermore, we also found identical staining for positivity between most neoglycoproteins and their corresponding lectins, though the staining intensity of neoglycoproteins was weaker. This suggests that neoglycoproteins may be useful markers to replace their plant lectins.

Comparative study of the expression of DNA mismatch repair genes, the adenomatous polyposis coli gene and growth arrest DNA damage genes in melanoma recurrences and metastases.

The main goal of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2), the adenomatous polyposis coli (APC) gene and growth arrest DNA damage inducible (GADD) genes (GADD34, GADD45 and GADD153) in the different stages of melanoma recurrences and metastases, and to identify any mutual consistencies in their expression pattern. All the cases of primary melanoma examined showed a reduced expression of DNA repair genes. These results demonstrate that disturbances of DNA repair begin in the early stages of melanoma. No significant differences were found in the expression of these markers between cutaneous melanomas and their recurrences and metastases (P> 0.05). Eighteen significant correlations between markers were found in the primary melanomas, and 10 significant correlations were observed in the first recurrences of melanoma. In contrast, 27 statistically significant relationships were demonstrated in metastatic lymph nodes. The different correlations found in primary and metastatic tumours confirmed the hypothetical difference in marker interaction in the diagnostic groups investigated. Our results suggest that DNA repair genes may play an important role in the recurrence and metastasis of melanomas.

Analysis of the DNA mismatch repair proteins expression in malignant melanomas.

The importance of properly functioning DNA mismatch repair has been shown in several tumour types both hereditary and sporadic, but not yet in malignant melanomas. The aim of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2) in primary melanomas and to define their possible prognostic impact in 106 primary melanomas. MLH1 was found in 64 and MSH2 in 61 out of 106 melanomas. PMS1 and PMS2 proteins were present in 69 and 67 tumours, respectively. Loss of the expression of DNA mismatch repair proteins correlated with the increase of Clark levels. Cox regression analysis demonstrated some prognostic significance for PMS1 (forward p = 0.0018 and backward selections p = 0.0277), MLH1 (only forward selection p = 0.0081) and MSH2 (only backward selection p = 0.0115).

Graft-versus-host disease-like immunophenotype and apoptotic keratinocyte death in paraneoplastic pemphigus.

Paraneoplastic pemphigus (PP) is an autoimmune disease, which is frequently associated with non-Hodgkin's lymphoma. Autoantibodies against components of the cytoplasmic plaque of epithelial desmosomes are usually present in the sera and are believed to play a major pathogenic part in acantholysis and suprabasal epidermal blistering. However, another typical histological feature of PP, interface dermatitis with keratinocyte dyskeratosis, is shared with skin diseases that involve epithelial damage mediated by T cells. Here, we present the detailed characterization of the cutaneous T-cell response in a patient with PP and demonstrate a selective epidermal accumulation of activated CD8+ T cells together with an increased local production of interferon-gamma and tumour necrosis factor-alpha, and a strong expression of HLA-DR and ICAM-1 on keratinocytes. Apoptosis was identified as a key mechanism of keratinocyte death, and appeared independent of the FAS/FAS ligand (FAS-L) pathway, as epidermal expression of FAS was not increased compared with normal skin, and FAS-L was undetectable on the protein and mRNA level. Triple therapy with high-dose corticosteroids, cyclophosphamide and intravenous immunoglobulins reduced levels of pemphigus-like autoantibodies and reversed the cutaneous inflammatory reaction leading to long-standing clinical remission. Our findings support the concept of a major contribution of cytotoxic T lymphocytes to the immunopathology of paraneoplastic pemphigus.