Abstract
The DNA aneuploidy and proliferative activity have been previously shown to carry a clinical significance in Iymphoid malignancies. However, the application of these parameters is complicated by methodological difficulties. Initially, this study focused on the methodological assessment of image cytometry (ICM) when applied to hematological material. Additionally, the utility of confocal scanning laser microscopy for the quantification of the DNA content in routinely processed thick (30-40µm) was investigated. The presented 3-dimensional confocal ICM method makes it possible to avoid the artifacts caused by measurements of incomplete cut nuclei and allows reconstruction of the tissue in three dimensions.
The conventional ICM was applied to study DNA ploidy and the proliferative activity in morphologically defined cell subpopulations of acute Iymphoblastic leukemia (ALL), non-Hodgkin's Iymphoma (NHL) and Hodgkin's disease (HD). The cell proliferation was also assessed in the histological material with Ki-67 and anti-PCNA antibodies. The comparison of PCNA stainings on histological material to Ki-67 counts from cytological material showed that FNABs represent alternative material for estimation of proliferative activity in Iymph nodes. In childhood ALL ICM provided information complementary to results of conventional karyotyping. In a substantial fraction of follicle center NHLs centroblastic population was DNA tetraploid. The fluorescent in situ hybridization analysis of centroblasts from those Iymphomas indicated that neoplastic cells underwent polyploidization. In low grade follicle center NHLs high proliferative activity and in addition high apoptotic fractions correlated to achievement of complete remission.
On the basis of results from this ICM study a model of DNA ploidy evolution in follicle center NHLs is proposed. According to this model, the DNA content changes from DNA diploidy through DNA diploidy-tetraploidy, DNA tetraploidy to DNA aneuploidy. DNA tetraploid low-grade follicle center Iymphomas are prone to transformation to high-grade Iymphoma. The DNA ploidy, the proliferative activity and the expression of tumor suppressor gene product p53 and p21waf1/cip1 were investigated in a series of T-cell rich B-cell non-Hodgkin's Iymphoma simulating Hodgkin's disease. This Iymphoma resembles high grade B-cell NHL with regard the DNA content and p53 positivity. The expression of p21waf1/cip1 protein was almost exclusively found in highly DNA aneuploid Iymphomas with nuclear atypia such as Hodgkin's disease and T-cell rich B-cell non-Hodgkin's Iymphomas simulating Hodgkin's disease.
