[Multilayer analysis of signal transduction and cell cycle control in GIST. Identifying new interaction partners with differential regulation]. / Multilayer-Analyse der Signaltransduktion und Zellzykluskontrolle in GIST. Identifizierung neuer Interaktionspartner mit differenzieller Regulation.

To identify new interactions as well as diagnostically, prognostically and therapeutically relevant differences in the regulation of gene expression in gastrointestinal stromal tumors (GISTs), we analyzed the methylation status, mRNA expression, microRNA expression, protein expression and protein phosphorylation in parallel in identical tumor tissue samples. The data were analyzed in a multilayer approach and were correlated to each other and to clinico-pathological parameters. Differentially regulated genes were mapped to signal transduction pathways which are already known to play a major role in GISTs. A functionally orientated overview of the different data layers was constructed, which enabled new insights into gene regulation in GISTs.

Angiogenesis and lymphangiogenesis are downregulated in primary breast cancer.

BACKGROUND: Angiogenesis and lymphangiogenesis are considered to play key roles in tumour growth, progression and metastasis. However, targeting tumour angiogenesis in clinical trials showed only modest efficacy. We therefore scrutinised the concept of tumour angiogenesis and lymphangiogenesis by analysing the expression of crucial markers involved in these processes in primary breast cancer. METHODS: We analysed the expression of angiogenic, lymphangiogenic or antiangiogenic factors, their respective receptors and specific markers for endothelial and lymphendothelial cells by quantitative real-time RT-PCR in primary breast cancer and compared the expression profiles to non-cancerous, tumour-adjacent tissues and breast tissues from healthy women. RESULTS: We found decreased mRNA amounts of major angiogenic and lymphangiogenic factors in tumour compared to healthy tissues, whereas antiangiogenic factors were upregulated. Concomitantly, angiogenic and lymphangiogenic receptors were downregulated in breast tumours. This antiangiogenic, antilymphangiogenic microenvironment was even more pronounced in aggressive tumours and accompanied by reduced amounts of endothelial and lymphatic endothelial cell markers. CONCLUSION: Primary breast tumours are not a site of highly active angiogenesis and lymphangiogenesis. Selection for tumour cells that survive with minimal vascular supply may account for this observation in clinical apparent tumours.

Increased KIT signalling with up-regulation of cyclin D correlates to accelerated proliferation and shorter disease-free survival in gastrointestinal stromal tumours (GISTs) with KIT exon 11 deletions.

Gastrointestinal stromal tumours (GISTs) with deletions in KIT exon 11 are characterized by higher proliferation rates and shorter disease-free survival times, compared to GISTs with KIT exon 11 point mutations. Up-regulation of cyclin D is a crucial event for entry into the G1 phase of the cell cycle, and links mitogenic signalling to cell proliferation. Signalling from activated KIT to cyclin D is directed through the RAS/RAF/ERK, PI3K/AKT/mTOR/EIF4E, and JAK/STATs cascades. ERK and STATs initiate mRNA transcription of cyclin D, whereas EIF4E activation leads to increased translation efficiency and reduced degradation of cyclin D protein. The aim of the current study was to analyse the mRNA and protein expression as well as protein phosphorylation of central hubs of these signalling cascades in primary GISTs, to evaluate whether tumours with KIT exon 11 deletions and point mutations differently utilize these pathways. GISTs with KIT exon 11 deletions had significantly higher mitotic counts, higher proliferation rates, and shorter disease-free survival times. In line with this, they had significantly higher expression of cyclin D on the mRNA and protein level. Furthermore, there was a significantly higher amount of phosphorylated ERK1/2, and a higher protein amount of STAT3, mTOR, and EIF4E. PI3K and phosphorylated AKT were also up-regulated, but this was not significant. Ultimately, GISTs with KIT exon 11 deletions had significantly higher phosphorylation of the central negative cell-cycle regulator RB. Phosphorylation of RB is accomplished by activated cyclin D/CDK4/6 complex, and marks a central event in the release of the cell cycle. Altogether, these observations suggest increased KIT signalling with up-regulation of cyclin D as the basis for the unfavourable clinical course in GISTs with KIT exon 11 deletions.

Characterization of a newly established uterine carcinosarcoma cell line featuring the sarcomatous phenotype of the tumor in vitro.

We describe the newly established cell line CS-99 derived from a uterine carcinosarcoma retaining features of the sarcomatous phenotype in vitro. CS-99 cells exhibit a mesenchymal morphology with predominantly spindle-shaped cells at nonconfluence turning to pleomorphic appearance at confluence. The mesenchymal phenotype was evidenced immunohistochemically by strong vimentin and moderate SM-actin, which was similar to the sarcomatous component of the primary tumor. P53 was overexpressed in a subset of CS-99 cells. Epithelial membrane antigen was moderately expressed whereas other markers including pan CK, CK 5/6, CK 34, epidermal growth factor receptor, desmin, carcinoembryonic antigen, S100, KIT, ERBB2, and the hormone receptors, estrogen receptor and progesterone receptor revealed either weak or no specific staining in CS-99 cells. High self-renewal capacity corresponded to the population doubling time of 23 h in high passage. CS-99 cells were able to develop three-dimensional tumor spheroids in vitro. Cytogenetic analysis and multicolor fluorescence in situ hybridization of CS-99 demonstrated an almost stable karyotype including numerical changes +8, +18, and +20 and translocations, amongst others der(1)t(1;2), der(1)t(1;7), der(2)t(2;19), der(5)t(5;8), and der(5)t(5;14). Taken together, the cell line CS-99 exhibits strong growths dynamics and a complex but stable karyotype in higher passages, and can be further a useful in vitro model system for studying tumor biology of carcinosarcomas.

Morphological and immunohistochemical characterization of isolated tumor cells by p53 status in gastrointestinal tumors.

BACKGROUND AND AIMS: Isolated tumor cells (ITCs) in cancer patients are retrieved mostly using immunohistochemistry with antibodies directed against antiepithelial antigens (for example Ber-EP4), which are supposed not to be present in metastatic-free tissue. To date, there has been ongoing controversy whether those cells have biologic significance and are linked with tumor progression and impaired patient's prognosis. Therefore, the aim of this study was to further characterize Ber-EP4-positive cells in various tissues, with special emphasis on their tumorigenic origin. MATERIALS AND METHODS: The frequency and prognostic impact of ITCs in lymph nodes displayed by means of monoclonal antibody Ber-EP4 were evaluated in retrospective (n = 292) and prospective (n = 100) collectives of various gastrointestinal carcinomas free of metastatic disease in conventional histopathology (pN0). Furthermore, the frequency of ITCs in the peritoneal cavity and bone marrow was analyzed in case of absence of overt distant metastasis (pM0) in the prospective collective. Ber-EP4-immunoreactive cells were further characterized for tumorigenic origin using morphological criteria and immunohistochemical double staining for Ber-EP4 and p53. RESULTS: Ber-EP4-positive cells could be revealed in lymph nodes in 44.3% of pN0-gastrointestinal carcinomas, in the peritoneal cavity in 19%, and in the bone marrow in 10%. In lymph nodes, BerEP4-immunoreactive cells exhibited a metastatic-atypical morphology in 59%; however, it was always typical for true tumor cells in the peritoneal cavity or bone marrow. The cumulative 5-year survival rate was adversely affected by Ber-EP4-immunoreactive cells in uni- and multivariate analysis, irrespective of the underlying cell morphology (68% for Ber-EP4 negative, 41% for Ber-EP4 positive with atypical and typical morphology each). In the case of a p53-positive primary tumor, 70% of the corresponding ITCs also overexpressed p53, while the remainder was deemed p53 negative (p = 0.002). CONCLUSION: ITCs detected by the antiepithelial antibody Ber-EP4 are present in a substantial proportion of apparently tumor-free lymph nodes. These cells impair patients' prognoses, irrespective of the underlying cell morphology. As approximately one third of Ber-EP4-positive cells in p53-positive primary tumors do not overexpress p53; their true tumorigenic origin needs to be further investigated.

Parvovirus B19 detected in Rosai-Dorfman disease in nodal and extranodal manifestations.

Sinus histiocytosis with massive lymphadenopathy (SHML), also designated as Rosai-Dorfman disease (RDD), is a rare benign reactive lymphoproliferative disorder. It is defined by a characteristic histopathology with sinus histiocytosis and haemophagocytosis known as emperipolesis. In histiocytes S100 is strongly expressed, whereas CD1a staining typically is negative. The disease mainly manifests at a single lymph node; however, multilocular and extranodal affection can occur. Causative infectious agents, and virus infections in particular, have repeatedly been suspected, although until now the origin of the disease has been unclear. Four cases of RDD (two nodal sites and two extranodal upper respiratory tract sites) were analysed for parvovirus B19 (B19) infection by immunohistochemistry to detect B19 capsid proteins VP1/VP2. In all the four cases, huge numbers of B19-positive cells were partly detected. The positive cells were identified either as lymphocytes or, in one extranodal case, also as respiratory epithelial cells. This is the first report of B19 infection in RDD tissue, indicating that B19 may be associated with the pathogenesis of SHML.

Distal nephron renal tumors: microsatellite allelotype.

Tumors of varying malignant potential arise from the complex epithelial lining of the nephron. Although the molecular characteristics of renal clear cell carcinomas, which arise from the proximal tubule, have been studied, little is known about tumors that develop from other parts of the renal tubular system. To elucidate common molecular lesions that may contribute to the development or progression of nonproximal tubule renal tumors, we performed a detailed microsatellite allelotype of lesions thought to arise from the renal collecting duct. Eighteen collecting duct carcinomas (CDCs) and 13 renal oncocytomas were studied using highly informative microsatellite markers on all autosomal arms. Loss of heterozygosity (LOH) was identified on multiple chromosomal arms in CDCs and renal oncocytomas. Microsatellite analysis revealed LOH of 1q in 57% of informative CDCs. LOH was also observed on arms 6p (45%), 8p (40%), and 21q (40%). In renal oncocytomas, LOH of 1q occurred in approximately 30% of tumors, but 1p LOH was observed in 57% of informative cases analyzed. High levels of LOH were also observed on arms 8p, 14q, 19q, and 21q in the oncocytomas studied. Loss of chromosomal arm 3p was infrequent in both tumor types. Our results suggest that the molecular events that contribute to the development of distal nephron tumors are distinct from those associated with the etiology of proximal tubule renal cancers.

Prognostic impacts of cytogenetic findings in clear cell renal cell carcinoma: gain of 5q31-qter predicts a distinct clinical phenotype with favorable prognosis.

To evaluate the prognostic significance of cytogenetic findings in clear cell renal cell carcinoma (RCC), cytogenetic results of 118 primary RCCs were evaluated in relation to classical indicators of prognosis and overall survival. Losses in 3p (98.3%) were most prevalent and included 32 (27.6%) monosomies of chromosome 3 and 84 (72.4%) structural aberrations involving 3p, of which 36 were unbalanced translocations, der(3)t(3;5)(p11-p22;q13-q31), resulting in duplication of 5q sequences. Patients with gain of 5q31-qter resulting from either polysomies or structural rearrangements of 5q, the most frequent of which was der(3)t(3;5), had a significantly better outcome than those without this aberration (P = 0.001). There was no association between gain of 5q or der(3)t(3;5) and any of the well-known variables for prognosis, including low versus high clinical stage and grade of malignancy. Among additional chromosomal aberrations, loss of chromosome 9/9p was associated with distant metastasis at diagnosis (P = 0.006). The data indicate that gain of 5q identifies a clinically favorable cytogenetic variant of clear cell RCC and demonstrate the impact of specific chromosome aberrations as additional prognostic indicators in clear cell RCC.

Deformation-induced endothelin B receptor-mediated smooth muscle cell apoptosis is matrix-dependent.

To maintain normal blood flow, pressure overload in both arteries and veins requires a structural adaptation of the vessel wall (remodelling) that involves smooth muscle cell (SMC) hypertrophy and/or hyperplasia. Due to its potent vasoconstrictor and growth-promoting effects, endothelin-1 (ET-1) is a likely candidate to initiate and/or promote remodelling in blood vessels exposed to a chronic increase in blood pressure. To test this hypothesis, isolated segments of the rabbit carotid artery and jugular vein were perfused at different levels of intraluminal pressure. In both types of segments, pressure overload (160 and 20 mmHg, respectively) resulted in an increase in endothelial prepro-ET-1 and SMC endothelin B receptor (ETB-R) expression. Moreover, in pressurised segments from the carotid artery an ETB-R antagonist-sensitive increase in SMC apoptosis in the media was observed, while in the vein medial SMC started to proliferate. Isolated SMC from these rabbit blood vessels as well as from the aorta and vena cava of the rat, when cultured on a collagen or laminin matrix, uniformly revealed an ETB-R-mediated increase in apoptosis upon exposure to mechanical deformation plus exogenous ET-1 (10 nmol/L). However, when grown on a fibronectin matrix, the cultured SMC did not respond with an increase in apoptosis under otherwise identical experimental conditions. These findings suggest that deformation-induced activation of the endothelin system in the vessel wall not only plays a crucial role in remodelling, but that the structural components of the vessel wall, in particular the cell-matrix interaction, determine how SMC respond phenotypically to these changes in gene expression.

Simultaneous tumour-like, atypical basal cell hyperplasia and acinar adenocarcinoma of the prostate: a comparative morphological and genetic approach.

Basal cell tumours of the prostatic gland are rare, and the classification is difficult. In the present case report, a large, tumour-like proliferation of atypical basaloid cells was found incidentally in a prostatectomy specimen that otherwise contained a conventional acinar adenocarcinoma. The basaloid cells displayed a solid or adenoid-cystic growth pattern and strongly expressed high-molecular-weight cytokeratins and bcl-2. A high Ki-67 index was recorded within the atypical basaloid cells, by far exceeding the one counted in the conventional adenocarcinoma. However, there were no definite criteria for a malignant behaviour of the basal cell tumour. Comparative genomic hybridisation from microdissected tumour cells yielded losses at the short arms of chromosomes 8 and 12 in the conventional adenocarcinoma and a normal karyotype in the basal cell tumour. The pathological findings favoured the diagnosis of an atypical basal cell hyperplasia.

Expression of the sodium iodide symporter in differentiated thyroid cancer: clinical evidence.

AIM: Molecular analysis of the expression of the sodium iodide symporter (NIS) in 32 patients with differentiated thyroid cancer (DTC) and correlation with scintigraphic findings ((131)I,(123)I) in 19 (59.4%) of them. PATIENTS, METHODS: NIS expression of 27 primary tumours, 13 lymphnodes and 18 distant metastases was determined by immunostaining using a murine monoclonal anti-NIS-antibody. NIS expression and radionuclide uptake of metastases were analysed by a semiquantitative visual score. Patients were divided into two subgroups: Group 1 (n = 8 patients): indirect correlation of radioiodine uptake (RIU) of subsequent metastases with NIS expression of 7 primary tumours and 3 metastases; Group 2 (n=11 patients): direct correlation of radionuclide uptake with NIS expression of 19 metastases which were excised after imaging. RESULTS: 49 of 58 specimens (84.5%) were NIS-positive. A preserved NIS-expression was found in 12 primary tumours and 8 of 10 (80%) synchrone and 6 of 7 (85.7%) metachrone metastases. Group 1 revealed a 100% positive predictive value (PPV) of a preserved NIS expression in the primary tumour regarding radioiodine uptake in metastases while a lack of NIS expression in the primary tumor did not reliable predict a loss of the metastases' ability to concentrate radioiodine. In group 2, only 11 of 19 (57.9%) specimens showed a concordant NIS expression and RIU whereas in the remaining 8 cases without visible RIU NIS expression was still present. CONCLUSIONS: NIS expression of the primary tumour and metastases in DTC is usually well preserved. We found a positive correlation between NIS expression of the primary and metastatic tissue but could not identify such well correspondence between NIS expression and the RIU of subsequent metastases.

Prognosis factors in incisional hernia surgery: 25 years of experience.

BACKGROUND: Incisional hernia underwent a change from conventional techniques to mesh implantation. The relevance of different factors, like operative technique, mesh material, and patient-related parameters concerning the outcome following mesh repair, are still under debate. METHODS: In a comparative retrospective study of 421 incisional hernia operations on 348 patients, we investigated 241 Mayo procedures and 180 mesh repairs over a 25-year period. In addition to the quality of life following mesh implantation, the prognostic relevance of demographic, preoperative and intraoperative parameters, surgical technique, mesh material, and the surgeon's experience were analysed, both in a univariate and multivariate manner. RESULTS: With a mean follow-up of 9.7+/-8.8 years, the total recurrence rate following Mayo overlap was 37%, in contrast to 15% after mesh implantation (P=0.001). Mesh size was the only significant prognostic factor concerning quality of life following mesh implantation, and 86% of the patients with mesh repair were satisfied. The complication rate was determined significantly by patients' risk factors, size of hernia, operative technique, and the surgeon's experience, whereas the rate of recurrences was significantly influenced by the parameters obesity (BMI>25), size of hernia, and surgical experience. The recurrence rate decreased significantly with surgeon's experience-a minimum of 16 mesh repairs led to a recurrence rate of less than 10%. CONCLUSIONS: Only the mesh repair revealed acceptable recurrence rates with high patient comfort. From a surgical point of view, the most important prognostic factor following mesh repair is the surgeon's experience.

[Significance of occult lymphatic tumor spread in pancreatic cancer]. / Okkulte lymphatische Tumorzelldisseminierung beim Pankreaskarzinom.

PURPOSE: The aim of this study was to determine the frequency and effect on prognosis of occult tumor cells in regional lymph nodes judged to be tumor-free in conventional histopathology of pancreatic cancer patients. PATIENTS AND METHODS: Among 115 patients who underwent pancreatic resection for pancreatic (n=84) or distal common bile duct malignancy (n=12) or carcinoma of the papilla (n=19), 48 (42%) were staged pN0. Archival paraffin blocks of 271 resected regional lymph nodes of 41 pN0 patients were reevaluated for occult tumor cells using monoclonal antibody Ber-EP4. Cases with or without isolated tumor cells were compared regarding the distribution of various clinicopathological factors. RESULTS: Of 41 pN0 patients, 16 (39%) exhibited single Ber-Ep4 immunoreactive cells or small cell clusters in at least one lymph node. The occurrence of occult tumor cells was not dependent on other clinicopathological factors such as pT stage, grading, or curative resection. However, those cells were encountered more frequently in common bile duct carcinomas (100%) than in pancreatic (36%) or papilla (20%) carcinomas (P=0.009). Occult tumor cells impaired prognosis significantly in uni- and multivariate analyses (estimated 5-year survival 53% for pN0((i-)) vs 10% for pN0((i+)) and 9% for pN1/N2; P=0.0047). CONCLUSION: Occult tumor cells are frequent in apparently tumor-free lymph nodes of pancreatic cancer patients and often overlooked in conventional histopathology. They are encountered even in limited stages of disease and they impair prognosis, which is comparable to that of patients with true lymphatic metastases. Occult tumor cells in lymph nodes of pancreatic cancer patients could be used to stratify adjuvant therapy.

[In-vitro study of the cellular response of human fibroblasts cultured on alloplastic hernia meshes. Influence of mesh material and structure]. / Hernienchirurgie: Wachstumsverhalten humaner Fibroblasten auf alloplastischen Kunststoffnetzen. In-vitro-Studie zum Einfluss des Materials und der Oberflächenstruktur.

BACKGROUND: The biocompatibility of meshes in hernia surgery seems to be influenced markedly by the amount of the selected material and its structure. Fibroblasts play a major key role during the process of mesh incorporation. This study was performed to investigate differences in cell morphology and proliferation of human fibroblasts cultured on different polypropylene meshes. METHODS: In the present in vitro study the cellular response of human fibroblasts was investigated by scanning electron microscopy (SEM), comparing three different polypropylene meshes: a newly constructed low-weight and microporous mesh (NK1), a low-weight and macroporous mesh with absorbable polyglactin filaments (Vypro), and a heavy-weight and microporous mesh (BiomeshP1). Human fibroblasts (1,5.10(5) cells) were incubated with the meshes (each 12 mm(2)) for 6 hours, 5 days, 2, 4, 6, and 12 weeks. Computer-assisted morphometry of the fibroblast/mesh surface ratio served to reflect the biological cell response. RESULTS: The Vypro mesh showed the significantly highest fibroblast density during the first 6 weeks, but cell growth was nearly exclusively limited to the polyglactin filaments. At 3 months, after reabsorption of the polyglactin, the fibroblast-coated polypropylene mesh surface was only 50% compared to NK1 and BiomeshP1. The morphologic aspect of the fibroblasts on the BiomeshP1 mesh was much more degenerative and unphysiological, compared to NK1 and Vypro, with isolated, single cells instead of a broad, connective growth. The BiomeshP1 showed a significantly higher fibroblast proliferation around the nodes of the mesh compared to the straight filaments. On the NK1 mesh fibroblasts exclusively proliferated on the filaments but not on the pressed mesh surface. CONCLUSIONS: The polymer surface and structure appears to be of major importance for the biocompatibility of meshes: human fibroblasts preferably grow on low-weight meshes, thin filaments, and mesh nodes. Heavy-weight meshes induce degenerative cell reactions. Polyglactin seems to further improve cell proliferation whereas a pressed mesh surface without pores hinders fibroblast growth.

mRNA expression of matrix metalloproteases and their inhibitors differs in subtypes of renal cell carcinomas.

Altered expression of matrix metalloproteases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteases (TIMPs), has been demonstrated in various tumour tissues. mRNA expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, MMP-12, MMP-14 and TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 30 renal cell carcinomas (RCC), as well as in the surrounding tissues. Expression of the MMPs was significantly stronger in the carcinomas than in non-malignant tissues. High levels were demonstrated particularly in clear cell RCCs (CC-RCC). Except for MMP-1, MMP expression in the papillary RCCs (P-RCC) was, for most MMPs, significantly lower. Expression of the TIMPs in malignant cells of both subtypes was weak, with the exception of TIMP-4 which was strongly expressed in the P-RCCs and downregulated in the CC-RCCs. The latter was correlated with chromosomal loss of 3p, harbouring the TIMP-4 gene locus. In conclusion, deregulated expression of the MMPs and TIMPs in RCCs differs according to histology, grade, size and cytogenetic characteristics, suggesting that MMP and TIMP expression patterns play an important role for the typical histomorphological features of RCC subtypes and their respective biological behaviour.

Cytogenetic findings in a metastasizing primary testicular chondrosarcoma.

The first case of a primary chondrosarcoma of the testis is reported. The testicular tumor, 2.5 cm in diameter, metastasized to the retroperitoneal lymph nodes in a 24-year-old male. Histologically, the testicular tumor and the lymph node metastases revealed a well-differentiated chondrosarcoma. Cytogenetic analysis of the retroperitoneal metastases showed a complex chromosomal aberration with two copies of the isochromosome of the short arm of chromosome 12. This is a specific chromosomal anomaly in testicular germ cell tumors. Thus, the cytogenetic analysis proved the germ cell origin of tumor cells as opposed to a possible paratesticular mesenchymal histogenesis. Moreover, the triploid modal chromosomal number of tumor cells confirmed the relationship between polyploidization and a high degree of differentiation in germ cell tumors. These findings stress the clinical relevance of the cytogenetic analysis in at least a subset of cytogenetically well-characterized solid tumors.

A novel management strategy of steroid-free immunosuppression after liver transplantation: efficacy and safety of tacrolimus and mycophenolate mofetil.

BACKGROUND: Corticosteroids have been used traditionally for immunosuppression after solid organ transplantation. The variety of modern immunosuppressive agents offers the chance to replace drugs with an unfavorable risk-benefit ratio. The objective of this prospective pilot study was to investigate a novel steroid-free immunosuppressive regimen after clinical liver transplantation. METHODS: 30 adult liver graft recipients were included in an intent-to-treat analysis. Dual induction immunosuppression consisted of tacrolimus and mycophenolate mofetil. Prophylactic steroids were not given. Efficacy and safety parameters analyzed were patient and graft survival, incidence and severity of rejection, and adverse events in correlation to immunosuppressive drug levels. RESULTS: Patient and graft survival at 2 years was 86.7 and 83.9%, respectively. Acute rejection occurred in 26.2%, and was associated with subtherapeutic tacrolimus blood levels and diarrhea. All rejections were completely reversible by temporary addition of steroids. Acute renal failure was seen in 10/30 patients, and was related to high tacrolimus blood levels together with primary liver graft dysfunction. 43% of all patients never received any steroids, and 73% were on a steroid-free maintenance regimen. CONCLUSIONS: These results confirm that corticosteroids can be completely avoided from the beginning after liver transplantation. Double drug immunosuppression with tacrolimus and mycophenolate mofetil is effective and safe in terms of patient and graft survival as well as incidence and severity of rejection. In order to avoid under- or over-immunosuppression, which may be caused by impaired absorption or metabolism, close drug monitoring is advised.

p53 expression in Wilms' tumor: a possible role as prognostic factor.

Although a correlation between anaplasia and mutations of the p53 tumor suppressor gene has been found in Wilms' tumor (WT) a prognostic significance of p53 in WT remains largely unresolved. The goal of this study was to obtain a better understanding of the role of p53 expression in WT. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tumor tissues from 21 patients treated in our clinic between 1984 and 1996. Eight patients presented with stage I, six with stage II, two with stage III, four with stage IV and one patient with stage V disease. According to the presence of anaplasia, four cases were categorized as of unfavorable histology based on the criteria of the National Wilms' Tumor Study Group. Seven out of 21 WTs were positive for p53. One out of the eight patients with stage I and one out of the six patients with stage II disease scored positive for p53 as were 2/2 patients with stage III and 3/4 patients with stage IV disease. Four tumors scored positive for anaplasia (one stage I, one stage II and two stage IV disease) and all four belonged to the p53 positive group. Three of these patients died of progressive disease. Immunopositivity in general was focal in the blastemal and epithelial parts of the tumors, with differences in intensity of staining ranging from moderate to strong. Positivity in the stromal components was restricted to single cells. Statistical analysis revealed significant correlations of p53 expression to anaplasia and to survival, respectively. The association of p53 expression to tumor stage was of borderline significance. To support immunohistochemistry, we performed PCR/SSCP and DNA sequence analyses on two cases, one whose immunopositivity suggested a mutated p53, and another case which was immunonegative. A CGG --> TGG base change in codon 282 of exon 8 was found in the immunopositive tumor. In conclusion, p53 may be of prognostic relevance for poor outcome being present in close association with unfavorable histology of WTs.

Differential expression of the lung resistance-related protein/major vault protein in the histological compartments of nephroblastomas.

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Some tumors, however, fail to respond to therapy and those patients have a poor prognosis. In a search for molecular markers of drug resistance, we investigated the expression of lung resistance protein (LRP) in tissue samples from 32 children with nephroblastoma by means of immunohistochemistry. LRP is a human major vault protein (MVP) and is associated with multidrug resistance of tumors. LRP/MVP expression was found in the blastemal and epithelial compartments but to a significantly lesser extent in the stromal compartment of Wilms' tumors. Expression was generally heterogeneous with respect to staining intensity and percentage of positive cells. We found significant relationships between LRP/MVP expression and chemotherapeutic pre-treatment of tumors and tumor stage. The immunohistochemical results were validated with a real-time RT-PCR technique and a significant association between protein and mRNA expression was observed.

Differential expression of the multidrug resistance-related protein MRP1 in the histological compartments of nephroblastomas.

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Some tumors, however, fail to respond to therapy and those patients have a poor prognosis. In a search for prognostic markers, we investigated the expression of the multidrug resistance-related protein 1 (MRP1) in 32 nephroblastomas by means of immunohistochemistry. The immunohistochemical results were validated with a real-time RT-PCR technique. MRP1 expression was heterogeneous and predominantly found in the blastemal and epithelial compartments compared to the stromal elements of nephroblastomas. We found significant relationships of MRP1 expression to survival of patients and to expression of p53, HSP70, and LRP/MVP. The relationship between MRP1 and p53 expression is a clue that the transcriptional control of MRP1 by p53 reported for other tumor types may also take place in nephroblastomas. The correlation of MRP1 to other drug resistance genes, e.g. HSP70 and LRP/MVP in nephroblastomas indicates that the co-expression of different drug resistance genes may be under a common regulation of still unknown transcription factors.