Axel Niendorf

Esophageal Cancer: The Mode of Lymphatic Tumor Cell Spread and Its Prognostic Significance

PURPOSE: Data on skip metastases and their significance are lacking for esophageal cancer. This issue is important to determine the extent of lymphadenectomy for esophageal resection. In this study we examined the lymphatic spread in esophageal cancer by routine histopathology and by immunohistochemistry. PATIENTS AND METHODS: A total of 1,584 resected lymph nodes were obtained from 86 patients with resected esophageal carcinoma and evaluated by routine histopathology. Additionally, frozen tissue sections of 540 lymph nodes classified as tumor-free by routine histopathology were screened for micrometastases by immunohistochemistry with the monoclonal antibody Ber-EP4. The lymph nodes were mapped according to the mapping scheme of the American Thoracic Society modified by Casson et al. RESULTS: Forty-four patients (51%) had pN1 disease, and 61 patients (71%) harbored lymphatic micrometastases detected by immunohistochemistry. Skip metastases detected by routine histopathology were present in 34% of pN1 pat...

Early Lymphatic Tumor Cell Dissemination in Pancreatic Cancer : Frequency and Prognostic Significance

Tumor relapse occurs frequently in patients with ductal pancreatic head cancer despite the absence of residual tumor detectable at primary surgery. Therefore it has to be assumed that current tumor staging procedures fail to detect minimal amounts of disseminated tumor cells present in secondary organs, which might be the seed for subsequent meta-static relapse. We evaluated lymph nodes from 18 patients without overt metastases who had undergone radical tumor resection (R0 resection). Lymph nodes judged as “tumor-free” by routine histopathology were further examined for the presence of single tumor cells using immunohistochemistry with the antiepithelial monoclonal antibody Ber-EP4. Sixteen of 37 “tumor-free” lymph nodes (43.2%), obtained from 13 of 18 patients (72.2%), displayed Ber-EP4+ tumor cells. Twelve of these 18 patients presented at pT2 stage. Nine of 12 patients (75%) staged as pN0 had these cells. Two of six pN1 patients had no Ber-EP4+ in histopathologically tumor-free lymph nodes. Using multivariate Coxs regression analysis, the presence of Ber-EP4+ cells in “tumor-free” lymph nodes was an independent factor for a significantly reduced relapse-free survival (p = 0.006) and overall survival (p = 0.01). Remarkably, all patients who were restaged as lymph node negative by both histopathology and immunohistochemistry survived the observation period without recurrence. The frequent occurrence of disseminated tumor cells in patients with pancreatic cancer and their prognostic impact support the need for a refined staging system of excised lymph nodes, which should include immunohistochemical examination. Thus patients with a minimal residual tumor load who might benefit from an adjuvant therapy could be selected.

Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n = 74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.

Expression and prognostic significance of immunoregulatory molecules in esophageal cancer

Major histocompatibility complex molecules (HLA), the co‐stimulatory molecule B7 and the intercellular adhesion molecule 1 (ICAM‐1) are key molecules involved in T cell‐mediated immune surveillance. We aimed at assessing the expression pattern of these immunoregulatory molecules on primary esophageal carcinomas and evaluating their prognostic significance. Representative samples of primary tumors were obtained from 53 patients who had undergone radical en bloc esophagectomy without residual tumor. Cryostat sections of these tumors were stained with monoclonal antibodies (MAbs) directed against either HLA class I, HLA class II, B7 or ICAM‐1. The median follow‐up was 19 months (range, 6–43). We found that HLA class I expression was deficient on 27 tumors, while a significant neo‐expression of HLA class II, B7 and ICAM‐1 (≥25% positive tumor cells) was observed on 17, 29 and 25, tumors, respectively. Kaplan‐Meier analyses revealed a significant beneficial influence on relapse‐free survival for patients with tumors expressing HLA class I, HLA class II and B7. Coxs regression analyses demonstrated that co‐expression of HLA class I and ICAM‐1 was a significant and independent predictor of a reduced risk of developing tumor recurrence, whereas expression of ICAM‐1 on HLA class I negative tumors was correlated with an increased risk of tumor relapse. Int. J. Cancer 74:582–587, 1997.© 1997 Wiley‐Liss, Inc.

Probucol inhibits not only the progression of atherosclerotic disease, but causes a different composition of atherosclerotic lesions in WHHL-rabbits

Watanabe heritable hyperlipidaemic (WHHL)-rabbits develop premature atherosclerosis due to an inborn defect of the low-density lipoprotein (LDL) receptor causing severe hypercholesterolaemia. Probucol, which possesses a lipid lowering and an antioxidative potency, has been shown to reduce the extent of atherosclerotic disease in this animal. The object of the present study was the detailed analysis of the cellular and non-cellular composition of atherosclerosic lesions in WHHL-rabbits treated with probucol when compared with untreated controls. In two independent sets of experiments, each consisting of one litter, a total number of 5 animals was fed a diet containing 1% (w/w) probucol. Four animals served as controls and 2 animals were sacrificed before treatment (at 2 and 4 months of age, respectively) to define the baseline level of the atherosclerotic disease. Morphometric analysis was employed in order to determine plaque area macroscopically by planimetry and plaque thickness and composition histologically, in 30 cross-sections of the aorta of each animal. In the group treated with probucol, a diminution of plaque area and thickness, as well as a decrease of foam cell and — especially in one experiment — necrotic content of atherosclerotic lesions, was observed. Plaques from aortas of animals treated with probucol consisted predominantly of smooth muscle cells and compact intercellular fibrous structures. Furthermore, as an additional characteristic feature of the “typical” probucol plaque, they usually lacked confluent necrotic cores. In comparison with untreated animals, there was also a decrease in intracellular apolipoprotein B (apo B) as determined by immunohistochemistry. These data confirm the antiatherosclerotic potency of probucol in the WHHL-rabbit. Moreover, it was demonstrated that there is a different type of atherosclerosis present in the group treated with probucol. The mechanism behind these shifts may be based on the antioxidative property as well as on direct effects of probucol on cellular plaque components.

A new method for histological microdissection utilizing an ultrasonically oscillating needle: demonstrated by differential mRNA expression in human lung carcinoma tissue.

Molecular analysis of microdissected tissue samples is used for analyzing tissue heterogeneity of histological specimens. We have developed a rapid one-step microdissection technique, which was applied for the selective procurement of tissue areas down to a minimum of 10 cell profiles. The special features of our microdissection system consist of an ultrasonically oscillating needle and a piezo-driven micropipette. The validity of this technique is demonstrated in human lung large-cell carcinoma by real-time quantitative reverse transcriptase-polymerase chain reaction assays of vimentin, cyclin D1, and carcinoembryonic antigen after linear RNA amplification. mRNA expression values of microdissected samples scattered around those of bulk tumor tissue and showed differential mRNA expression between samples of tumor parenchyma and supportive stromal cells for vimentin and carcinoembryonic antigen as confirmed by immunohistochemistry. In conclusion, this procedure requires simple equipment, is easily performed, and delivers microdissected tissue samples of oligocellular clusters suitable for further molecular analysis.

Nuclear DNA content of borderline tumors of the ovary: correlation with histology and significance for prognosis

Scanning-DNA cytophotometry was applied to Feulgen stained sections of 22 borderline tumors of the ovary (BOT). The DNA content was related to conventional histology. In 11 cases clinical follow up for more than 5 years was available. The DNA measurements disclosed two subgroups in the group of BOT. One showed a nuclear DNA content not exceeding tetraploidy (4c) indicating proliferative activity without malignant change and a second one exhibited DNA values higher than 4c indicating malignant transformation. Correlation of histological evaluation with the DNA content revealed a good agreement in 15 cases. However, a discrepancy was found in 7 cases: either the histological evaluation aroused suspicion for malignant potential but the histogram showed DNA values not higher than 4c (n=4), or histology showed well differentiated lesions with an atypical histogram (n=3). Clinical monitoring revealed no recurrence or tumor spread in all but one case of the group of lesions with DNA values up to 4c, whereas in the group with atypical DNA histograms (DNA values>4c) relapse appeared in 6 out of 7 cases. The results suggest that DNA analysis has prognostic significance for BOT.

Effect of continuous vs intermittent application of 3-OH-tamoxifen or tamoxifen on the proliferation of the human breast cancer cell line MCF-7 M1

SummaryThe antiproliferative potency of 5x10-7M tamoxifen (TAM) and 3-hydroxytamoxifen (3-OH-TAM) was investigated during continuous (8 days) or intermittent (2 h every 2nd or 3rd day, respectively) application to the oestrogen-receptor-positive, estradiol-sensitive human mammary carcinoma cell line MCF-7 M1, a variant of MCF-7 wild type. Growth modulation was evaluated in parallel by counting cells and by measuring DNA content. Continuous incubation resulted in a growth inhibition to 21.8±3.2% by 3-OH-TAM and to 39.5±4.8% by TAM when compared with control cultures defined as 100%. Intermittent addition induced a growth reduction to 23.0±2.1% by 3-OH-TAM and to 41.2±2.4% by TAM in relation to 100% controls. Addition of 3-OH-TAM for 2 h only at day 1 resulted in an inhibition to 70.3±3.2%, again in relation to 100% controls. When TAM was administered once for 2 h at day 1 it induced an inhibition to 79.0±4.9% at day 8. The in vitro results indicate that (a) at 5x10-7M3-OH-TAM has a better antiproliferative effectiveness than TAM, (b) the intermittent application is as effective as continuous application (no significant difference), and (c) the addition once a week reveals only a slight growth reduction after 8 days of culture.Application of the long-living TAM results in continuously high serum concentrations, which have been shown to create resistant cell clones. Compared to TAM the 3-OH metabolite has a considerably shorter half-life and its application in vivo reveals rise and fall of its serum concentrations. Since the presented data demonstrate that 3-OH-TAM is more potent than TAM and that the intermittent application is as effective as the continuous form, interval therapy with 3-OH-TAM may slow down the process of acquiring resistance to antioestrogens.

Establishment of primary cell cultures: Experiences with 155 cell strains

SummaryCell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements (growth factors, hormones), and (4) attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.

Effects of low-dose pravastatin sodium on plasma cholesterol levels and aortic atherosclerosis of heterozygous WHHL rabbits fed a low cholesterol (0.03%) enriched diet for one year

The aim of this study was to evaluate the cholesterol-lowering and antiatherosclerotic effect of the HMG-CoA reductase inhibitor pravastatin sodium at a dosage comparable to human therapy. Twelve heterozygous WHHL rabbits (13 months old) were fed 100 g per day of a low cholesterol (0.03%) enriched diet for 12 months. Six of these animals also received pravastatin sodium at a daily dose of 1 mg/kg body weight (verum group). In the verum group, total plasma cholesterol levels were lower by 47%(P < 0.05) and relative aortic plaque volume (% ratio of total plaque volume to the aortic lumen) was reduced by 78% (P < 0.05), when compared to the control group. Plaque composition was analysed at 30 cross-sectional levels of the entire aortic wall using a grid window. Compared to the control group, the plaque type, in terms of architecture and composition, was altered as follows: lesions in the verum group had no confluent atheromatous cores and showed a pattern of a diffuse mixture of the main plaque components with a decreased relative content of necrosis (-44%) and an increased relative content of smooth muscle cells (+19%), whereas the relative content of macrophage-derived foam cells and collagen were nearly unaffected. Furthermore, a similar plaque volume and type was observed in animals with comparable cholesterol profiles. There was no histologic evidence for structurally damaging effects of pravastatin sodium on the arterial wall. We conclude that pravastatin sodium reduces total plasma cholesterol levels in this animal model, thereby leading to smaller plaques and a different plaque type.