p53 Mutations in carcinoma of the esophagus and gastroesophageal junction.

BACKGROUND: Recent studies suggested p53 mutations as a prognostic factor. Tumors of the esophagus and gastroesophageal (GE) junction show raising incidence with a general poor prognosis. METHODS: p53 Mutational spectra in 103 patients (68 squamous cell carcinoma/SCC and 35 adenocarcinoma/AC) were compared to clinical and pathologic data. RESULTS AND CONCLUSIONS: p53 Mutations were found in 26 of 68 SSC (38.2%) and in 12 of 35 AC (34.5%). We only found G > T transversions in smokers with SCC. The survival of patients was not affected by p53 mutational status. In our study, the frequency and mutational spectrum of mutant p53 is similar in both histological types without prognostic relevance.

The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.

The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins.

Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67.

The human antigen defined by the monoclonal antibody Ki-67, the 'Ki-67 protein', is an ubiquitously expressed human nuclear protein strictly associated with cell proliferation and is widely used in routine pathology as a 'proliferation marker' to measure the growth fraction in human tumours. In immunoblots of proteins from proliferating cells, Ki-67 detects two bands with the apparent molecular weights of 345 and 395 kDa. Recently we reported on the cloning and sequencing of the complete cDNA of the Ki-67 protein. We found two isoforms of cDNA with full lengths of 11.4 and 12.5 kb, respectively, likely formed by the alternative splicing of exon 7. The remarkable exon 13 at the 'centre' of this gene contains 16 homologous segments of 366 bp (Ki-67 repeats), each including a highly conserved new motif of 66 bp (Ki-67 motif). Computer analyses confirmed that the cDNAs encode for a new class of nuclear proteins. The complete gene locus of the Ki-67 protein, comprising a 74 bp 5' region and a 264 bp 3' region, has been sequenced and aligned to a continuous sequence of 29,965 bp length located on chromosome 10q25-ter. The gene is organized in 15 exons with sizes from 67 to 6845 bp and in 14 introns with sizes from 87 to 3569 bp. Three introns contain homologue copies of 'Alu-repeats'. Interestingly, the introns flanking the alternative spliced exon 7 are free of any consensus donor and acceptor splicing signal. All other intron-exon transitions contained a potential branch site. The complete 5' region including the first two exons represents a CpG-rich island. We found the transcription initiation site in exon two adjacent to the consensus sequence of a cap site. Upstream of this cap site no TATA- or CCAAT-box could be located, but downstream we found two remarkable directly repeated elements of 24 bp lengths each containing a TATA box in inverse orientation.

Expression of MDR1 mRNA and encoding P-glycoprotein in archival formalin-fixed paraffin-embedded gall bladder cancer tissues.

The aim of this study was to examine the expression of P-glycoprotein (Pgp) and MDR1 mRNA, in gall bladder carcinoma, a chemo-resistant tumour. 26 cases of gall bladder cancer and nine samples of normal gall bladder archival paraffin blocks were investigated for the presence of Pgp protein with immunohistochemistry (IHC) and MDR1 RNA by reverse transcription-polymerase chain reaction (RT-PCR). Monoclonal antibodies JSB-1 and UIC-2, recognising separate epitopes of Pgp, were used for IHC. For RT-PCR, total RNA was extracted from paraffin-embedded tissue. After RT, the samples were subjected to nested PCR (NPCR) using primers specific for the MDR1 gene, and evaluated by electrophoresis. In gall bladder carcinoma, the percentage of positive cases expressing Pgp (77% for JSB-1, 69% for UIC-2) and MDR1 mRNA (52%) was significantly higher than those in normal gall bladder. In earlier TNM stages Pgp and MDR1 mRNA were more frequently expressed (non-significant) than in advanced stages. The results of this study suggested that overexpression of MDR1 mRNA and Pgp in gall bladder carcinoma tissue probably is a very important reason why gall bladder cancer is generally not responsive to chemotherapy.

p53 and Bcl-2 as significant predictors of recurrence and survival in rectal cancer.

The aim of this study was to evaluate the prognostic value of p53 nuclear accumulation and Bcl-2 expression after curative surgery for rectal cancer. Immunohistochemistry was performed using monoclonal antibodies (MAb) (DO-1 for p53; anti-human Bcl-2 MAb, clone 124, for Bcl-2) on formalin-fixed, paraffin-embedded tissues of 160 rectal carcinomas (UICC stages I-III), and results were compared with data from the prospective registry of rectal cancer by univariate and multivariate logistic regression model focusing specifically on recurrence. Survival was calculated by the Kaplan-Meier method and proportional hazards model. p53 nuclear accumulation was documented in 39% (n=63) of tumours and was associated with a higher incidence of tumour progression (local or distant recurrence) and poorer disease-free survival (P<0.0001). Bcl-2 expression was detected in 29% (n=47), and was associated with longer disease-free survival and lower incidence of recurrence (P<0.0086). Multivariate logistic regression analysis demonstrated that gender (P=0.0136), UICC stage (P=0.0002), p53 expression (P=0.0002) and Bcl-2 expression (P=0. 0243) were independent factors predictive of recurrence. The proportional hazards model identified p53 (P=0.0009), UICC stage (P=0.0480), gender (P=0.0049), but not Bcl-2 (P=0.1503), as independently related to disease-free survival. Looking at the p53/Bcl-2 subgroups, the poorest prognosis was observed in the p53+/Bcl-2- subgroup, whereas patients whose tumours were p53-/Bcl-2+ had the best prognosis (P<0.0001). Immunohistochemical assessment of both p53 and Bcl-2 status may be valuable in predicting recurrence and survival after curative surgery for rectal cancer. Therefore, they play a role as prognostic factors in rectal cancer. p53 is a stronger predictor of prognosis than Bcl-2.

Expression of p53 and mdm2 mRNA and protein in colorectal carcinomas.

The aim of our study was to investigate the expression of p53 and mdm2 mRNA and protein in colorectal adenocarcinoma. For the detection of mRNA, 60 fresh frozen human tumour samples and 12 samples of corresponding normal tissue were examined. After total RNA extraction, reverse transcription (RT) was performed followed by cDNA amplification with specific primers using RT-polymerase chain reaction (PCR). Immunohistochemical detection of protein was examined in 81 formalin-fixed and paraffin-embedded human tumour specimens as well as 15 samples of adjacent normal colorectal tissue. p53 mRNA was detected in 80% (48/60) of the tumours and in 67% (8/12) of normal tissue samples; 87% (52/60) of tumours had mdm2 mRNA in contrast to only 17% (2/12) of normal tissue specimens. Nuclear p53 protein expression was observed in 52% (42/81) of the tumour specimens and in none of the 15 normal specimens, whereas mdm2 protein was found in the nucleus (31%, 25/81) and also in the cytoplasm (86%, 70/81) of tumour samples. In normal tissue, mdm2 protein expression was only observed in the cytoplasm (13%, 2/15) and not in the nucleus. There was a significant correlation between coexpression of p53 and mdm2 protein and the occurrence of lymph node metastases (P = 0.03) as well as between p53 protein expression and the occurrence of distant metastases (P = 0.007). Additionally, significant associations were found between p53 mRNA and p53 protein, p53 mRNA and mdm2 mRNA or protein, and also between mdm2 mRNA and mdm2 protein expression, supporting the existence of a regulatory mechanism involving p53 and mdm2.

Detection of Mycobacterium leprae by three-primer PCR.

Recently, polymerase chain reaction has been introduced for the species-specific assessment of Mycobacterium leprae (1). To avoid Southern blotting techniques using radioactively labelled oligonucleotide probes, the aim of this study was to establish a three primer-based single-step PCR technique. Using primers designed for this purpose we amplified a part of the gene encoding for the 16S ribosomal RNA of slowly growing mycobacteria. Due to the species-specific antisense primer a second, smaller fragment specific for M. leprae was amplified. Our results show that the employment of a second antisense primer in the PCR may be a substitution for Southern blot hybridization.

Mycobacterium leprae DNA content, cellular and cytokine patterns in skin lesions of leprosy patients undergoing multidrug therapy (MDT).

Skin biopsies from untreated and MDT-treated patients were examined for infiltrating cells and cells producing the cytokines TNF-alpha, IFN-gamma, and IL-1 beta using immunohistochemistry. Biopsy specimens from untreated tuberculoid leprosy patients were characterized by the presence of cells producing TNF-alpha, IFN-gamma, and IL-1 beta and of subepidermal Langerhans cells. These cells were rarely found or completely absent in biopsies of untreated lepromatous leprosy patients, but tended to increase under MDT. In a short-term therapy trial for three months with brodimoprim, dapsone, and rifampicin, 12 patients were monitored by follow-up biopsies. Semiquantitative PCR for mycobacterial DNA revealed two groups of patients: one group in which mycobacterial DNA in follow-up biopsies remained constant and a second group in which a decrease of mycobacterial DNA during therapy was noted. Immunophenotyping in these follow-up biopsies revealed that in the latter group IFN-gamma-positive cells and Langerhans cells were present and gamma delta T cell receptor-positive cells tended to decrease during therapy. In contrast, in patients whose mycobacterial DNA did not change during therapy, these phenotypical manifestations were not observed. We therefore, conclude that assessment of mycobacterial DNA in combination with phenotyping of infiltrating cells and determination of cytokine patterns may be useful tools in establishing criteria for the effectiveness and duration of MDT in patients with leprosy.

Assessment of the proliferation index in gastric carcinomas with the monoclonal antibody MIB 1.

Our study aimed to reveal whether the proliferation index of tumor cells, calculated with the monoclonal antibody (mAb) MIB1, is of prognostic relevance in patients with a gastric carcinoma and shows any correlation to well-known clinicopathological factors (TNM categories, stage, grade, Laurén type). We examined formalin-fixed, paraffin-embedded tissue blocks of samples from 94 patients, who underwent surgery for an adenocarcinoma of the stomach between 1988 and 1991. Specimens were immunohistochemically stained using the mAb MIB1 in combination with the alkaline-phosphatase/anti-(alkaline phosphatase) technique. The proliferation index (PI) was estimated in various areas of interest (tumor center and periphery and in lymph node metastases of compartments I and II), by always counting 200 tumor cells in three different high-power fields per specimen, and calculated as the percentage of MIB1-positive tumor cell nuclei relative to all tumor cell nuclei in the area examined. The total PI in the primary tumor was 47.2% and slightly higher in the center (49.1%) compared to the periphery (44.7%). Surprisingly in lymph node metastases the PI was lower than in the primary tumor (compartment I: 39.5%, compartment II: 33.6%). Tumors with distant metastases revealed a higher proliferative activity (55.1%) than tumors without (44.3%). The PI increased significantly from well to poorly differentiated carcinomas (P < 0.01), whereas the intestinal Laurén type showed a lower PI than the diffuse type. No difference in survival was found between patients with a median PI or less and those with a PI above the median (47.2%). Our results show that the proliferation index in gastric carcinomas has no prognostic relevance and therefore is of low clinical value.

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS: To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS: Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS: A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS: After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein).

AIMS--To elucidate the fine specificities of the antibodies MIB 1 and MIB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, JG-67-2a). METHODS--Different parts of the Ki-67 protein cDNA were expressed in Escherichia coli. Bacterial lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to nitrocellulose. Additionally different peptides were synthesised on a membrane support (SPOT-Blot). The immunoreactivity of the antibodies with the recombinant proteins and the immobilised synthetic peptides, respectively, was analysed. A competition enzyme linked immunosorbent assay (ELISA) using a soluble synthetic peptide was also performed. RESULTS--The epitopes of all antibodies tested were contained within the same region of seven amino acids. The antibodies MIB 1 and MIB 3 required the five amino acid sequence FKELF for binding, whereas Ki-67, JG-67-2a, MIB 5 and IND.64 detected the sequence FKEL. CONCLUSIONS--It is concluded that the amino acid sequence FKELF represents an immunodominant area of the Ki-67 protein and that there is no correlation between the ability to detect the Ki-67 protein in paraffin wax sections irradiated with microwaves and the epitopes recognised by the antibodies.

Vascular endothelial growth factor (VEGF)--a valuable serum tumour marker in patients with colorectal cancer?

INTRODUCTION: Neo-angiogenesis, of great importance for tumour growth and nutrition, is preferentially mediated by the cytokine vascular endothelial growth factor (VEGF), which has a direct effect on vascular endothelial cell proliferation and migration. This study was designed to clarify whether VEGF is a suitable tumour marker in sera of patients with a colorectal cancer, and whether VEGF concentrations in sera and tumour tissues are correlated with tumour extension (pTNM) and especially with tumour volume or size. Furthermore, the influence of VEGF levels on patients >> prognosis was examined. METHODS: VEGF serum concentrations of 122 patients with colorectal cancer and 65 controls were determined with an ELISA kit. Additionally, VEGF concentrations of tumour and normal tissue were measured in 38 patients using the same ELISA. RESULTS: Our results demonstrate that VEGF is not a suitable diagnostic tumour marker in patients with colorectal cancer due to its low sensitivity (36%). However, a combination of the serum tumour markers CEA and VEGF can significantly increase the pre-operative diagnostic sensitivity to 62%. VEGF serum levels differed significantly between patients (mean 438 pg/ml) and controls (mean 203 pg/ml), and also between tumour and normal tissue (984 vs 89 pg/mg protein). Serum concentration showed a significant correlation to tumour volume and size. Patients with VEGF serum levels greater than cut-off had a poorer prognosis than those less than or equal to cut-off. For this reason VEGF could be used as a predictor of patients >> outcome.

Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.

A decade of studies on the human nuclear antigen defined by monoclonal antibody Ki-67 (the "Ki-67 protein") has made it abundantly clear that this structure is strictly associated with human cell proliferation and that the expression of this protein can be used to assess the growth fraction of a given cell population. Until recently the Ki-67 protein was described as a nonhistone protein that is highly susceptible to protease treatment. We have isolated and sequenced cDNAs encoding for this antigen and found two isoforms of the full length cDNA of 11.5 and 12.5 kb, respectively, sequence and structure of which are thus far unique. The gene encoding the Ki-67 protein is organized in 15 exons and is localized on chromosome 10. The center of this gene is formed by an extraordinary 6845 bp exon containing 16 successively repeated homologous segments of 366 bp ("Ki-67 repeats"), each containing a highly conserved new motif of 66 bp ("Ki-67 motif"). The deduced peptide sequence of this central exon possess 10 ProGluSerThr (PEST) motifs which are associated with high turnover proteins such as other cell cycle-related proteins, oncogenes and transcription factors, etc. Like the latter proteins the Ki-67 antigen plays a pivotal role in maintaining cell proliferation because Ki-67 protein antisense oligonucleotides significantly inhibit 3H-thymidine incorporation in permanent human tumor cell lines in a dose-dependent manner.

[Demonstration of the angiogenic cytokine vascular endothelial growth factor (VEGF) by non radioactive in situ hybridization]. / Nachweis des angiogenen Zytokins Vascular Endothelial Growth Factor (VEGF) durch nicht radioaktive In-situ-Hybridisierung.

In tumor angiogenesis Vascular Endothelial Growth Factor (VEGF) has an important role due to its target cell specificity. It is expressed by the tumor and effects on a paracrine pathway. To increase the understanding of its regulation, it is necessary to identify those cells releasing VEGF. This can be done by in situ hybridization (ISH). In this paper we present a protocol for non-radioactive ISH for VEGF colonic tissue. With this protocol it is possible to perform hybridization within one day.

[Polypropylene synthetic mesh modifies growth of human cells in vitro. An experimental study]. / Kunststoffnetze aus Polypropylen beeinflussen das Wachstum humaner Zellen in vitro. Eine experimentelle Studie.

INTRODUCTION: The aim of our study was to investigate the influence of polypropylene meshes on the proliferation and apoptosis of human cell cultures in vitro. METHODS: Human fibroblasts and HeLa cells were incubated in different densities (10(4), 3.10(4), 10(5) cells/well) together with polypropylene meshes (Prolene, 2 x 2 cm) in six-well culture dishes over a 48-h period. Cells without meshes served as controls. Cells were spun on slides and stained with the monoclonal antibody MIB-1. To calculate the proliferation indices, stained nuclei were counted. Apoptotic indices were determined by flow cytometric analysis, using FITC-conjugated Annexin-V and propidiumjodide for staining. RESULTS: Fibroblasts showed only a slight reduction of the proliferation index (PI) from 64% (controls) to 60% (meshes). Increasing cell density leads to a decrease in the PI of both groups. The PI of HeLa cells was similar in mesh groups and controls and independent of cell density. The apoptotic index (AI) of fibroblasts was significantly higher in the mesh group (3.7%) in comparison with the controls (1.9%). The same was observed for HeLa cells (AI mesh group: 4.5%, AI controls: 1.2%). Furthermore, an increase of AI was found with increasing cell density in both cell lines. CONCLUSION: Whereas meshes did not influence the proliferation of the cell lines examined, they seem to have a marked influence on apoptosis, as a significant increase of AI was observed in the mesh group in contrast to controls.

[Expression of P-glycoprotein in benign and malignant gallbladder neoplasms].

OBJECTIVE: To evaluate the relationship between P-glycoprotein expression and anti-cancer drug resistance of gallbladder carcinoma and the use of P-glycoprotein as a biomarker of gallbladder carcinoma, the expression of P-glycoprotein was detected in benign and malignant gallbladder neoplasms and normal gallbladder tissues. METHODS: Alkaline phosphatase anti-alkaline phosphatase (APAAP) method was used to detect the expression of P-glycoprotein in different gallbladder tissues (gallbladder carcinoma, 26 cases; benign gallbladder neoplasm, 14 cases; and normal gallbladder tissue, 9 cases). The relationship between expression of P-glycoprotein, TNM stages and other clinical data of gallbladder carcinoma was also analyzed. RESULTS: Immunohistochemical staining with a monoclonal antibody JSB-1, P-glycoprotein was positive in 76.9% (20/26) of gallbladder carcinomas, in 35.7% (5/14) of benign gallbladder neoplasms and in 33.3% (3/9) of normal gallbladder tissues (P < 0.05). With another monoclonal antibody UIC-2, the positive rates were 69.2% (18/26), 21.4% (3/14) and 11.1% (1/9), respectively (P < 0.01). There was no significant correlation between P-glycoprotein expression and gallbladder carcinoma TNM staging. CONCLUSION: The results suggest that P-glycoprotein probably plays an important role in the poor response of gallbladdar carcinoma to chemotherapeutic agents. In addition, P-glycoprotein may be used a valuable biomarker of gallbladder carcinoma.

Autologous blood vessels engineered from peripheral blood sample.

OBJECTIVE: Although many efforts have been made to generate small-diameter (< or =5mm) vascular grafts by means of tissue engineering, improvement in patency and functionality still remains a great challenge. It is our hypothesis that to achieve long-term functionality and patency, not only the complete lining with endothelial cells but also full biocompatibility is essential. DESIGN: The aim was the development of a conduit from a scaffold and endothelial progenitor cells (EPC) separated from peripheral blood of a single donor. MATERIALS AND METHODS: EPC and a fibrin preparation were separated from porcine peripheral blood. Fibrin segments were generated seeded with EPC and were perfused in a bioreactor in vitro. RESULTS: From 100ml blood 12-15 cm long fibrin tubes were successfully generated lined with endothelial-like cells. Seeded tubes showed a remarkable elasticity and burst strength up to 90 mm mercury. CONCLUSIONS: Stable fibrin tubes were successfully generated completely lined with an endothelium-like monolayer from fibrin and EPC, both isolated from the same volume of blood. Although their stability is not those needed for arterial grafting, our results raise the hope, that with distinct improvements in future studies functional autologous vascular grafts could be engineered from the patient's own blood.

Influence of thymidylate synthase and p53 protein expression on clinical outcome in patients with colorectal cancer.

AIMS: Thymidylate synthase (TS) and tumor suppressor p53 are two proteins with an influence on tumor resistance to radio-chemotherapy that is well known. For this reason we tested the effect of TS and p53 expression on clinical outcome (tumor recurrence and survival) in patients after curative tumor resection, especially in patients who received adjuvant radio-chemotherapy. PATIENTS AND METHODS: A total of 120 patients with colorectal cancer were included in the study. A curative resection was possible in 83 patients, and 30 of this group received adjuvant therapy. For the immunohistochemical staining of tumor specimens, monoclonal antibody (mAb) TS 106 against TS and mAb DO-1 against p53 protein were used. TS positivity was defined as a moderate to high staining intensity in the cytoplasma of cells and p53 positivity as nuclear staining of tumor cells in >10% of these cells. RESULTS: Thymidylate synthase immunoreactivity was found in 59% of all cases and p53 staining in 51%. No relation between clinicopathological features and p53 expression was found in contrast to TS expression, where a highly significant association of TS-positive cases with tumor invasion (pT) was observed. Curatively resected patients with a TS-positive tumor developed tumor recurrence/distant metastases significantly more often than TS negative tumors. The same result was found when comparing p53-positive with p53-negative tumors and TS+/p53+ with TS-/p53- tumors. TS expression was highly significantly associated with poor survival and was the strongest independent prognostic factor in multivariate analysis, followed by lymph node status. CONCLUSION: Thymidylate synthase expression seems to be an independent prognostic factor and a possible predictor of tumor recurrence in patients with colorectal cancer.