Original Research Paper

One of the most important questions in clinical routine is to find out patients with good or worse prognosis to apply an optimal therapy scheme for each patient. In this study 58 patients with different neuroendocrine tumours of the lung were investigated. Histological sections were prepared with different stainings (MIB-1, AgNOR, Feulgen). By means of high resolution image cytometry stereological parameters were derived which are indicators for proliferation, ploidy and kinetics of the tumours. Cox regression analysis was calculated to test the significance of the parameters with regard to prognosis. The best parameter was MIB-1 which can easily be applied as a clinical standard staining and measurement.


INTRODUCTION
Lung cancer is in industrial countries the most frequent cause of death for men and women.The overall 5-year survival rate is only about 15%.One part of lung tumours are neuroendocrine tumours divided in subtypes with different malignant potential (benign or low-grade malignant tumours, called typical carcinoids (TC) and on the other side the high-grade malignant tumours, poorly differentiated of small (SCLC) or large cell type (LCLC).Between these tumour types, the welldifferentiated carcinoma with a lower grade of malignancy (WDNEC) take place (WHO, 1998).In clinical routine it is important to distinguish patients with better and worse prognosis.The aim of this study was to test the markers MIB-1, AgNOR and DNA distribution parameters, which are applied as different biological indicators of proliferation, ploidy and kinetics, with regard to the survival of patients and to the improvement of their therapy.

MATERIAL AND METHODS
In this study 32 cases of SCLC, 13 of WDNEC and 14 of TC with a follow-up time up to 7 years were collected.In Table 1 the complete clinical data set is shown.The tumour block was sliced into 4 µm thick sections from paraffin embedded tissue from routine and were stained afterwards according to MIB-1 (Fig. 1) (Böhm et al., 1996), AgNOR (Fig. 2) (Aubele et al., 1994a;Böhm et al., 1993) and Feulgen (Fig. 3) (Aubele et al., 1994b;Jütting et al., 1999).

Stereological parameters
( ) For evaluating the 'real' DNA distribution from sections with different thickness following formulae have to be taken into account with the assumption that the nuclei are nearly round objects: From the DNA distribution the mean value, 5cexceeding rate, entropy, 2c-deviation index, value of stemline peak, proliferation status and the euploid/ aneuploid status (Ploidy) were calculated (Schenck et al., 1997).In Fig. 4

STATISTICS
Stepwise Cox regression analysis was applied to search for features correlated with survival time (Lee, 1980).To demonstrate the results Kaplan-Meier curves for different strata are plotted.SAS and BMDP statistical packages were used.All statistical evaluations were done at 95% level.
r = radius of nucleus t = thickness of section IOD = integrated optical density (in extinction), normalization factor of lymphocytes = 1.6 due to the underestimation of IOD of the dark and condensed lymphocytes, then some typical DNA distributions for the different histological tumour types are shown.Entropy: Entropy describes the information content and disorder of a histogram.Its lowest value is reached if all DNA-values are found in the same channel of the histogram.Its maximum will be reached, if all measured cells are equally distributed over all channels.In this histogram feature the cells are weighed with the square of their distance to the normal DNA content of 2c.

Table 1 .
Clinical data of 58 tumour patients of the lung separately for each tumour type.