Multimodal image analysis of chronic leukemic lymphoproliferative disorders and the hypothesis of "single" and "multiple" programmed stops in the development of typical and atypical forms of leukemias and lymphomas.

The study consisted of morphometric analysis, assessment of the argyrophilic nucleolar organization region (AgNOR) characteristics, and image cytometry (ICM) in different tumor mass compartments: bone marrow (BM), peripheral blood (PB) and lymph nodes (LN) from patients with chronic leukemic lymphoproliferative disorders. A total of 71895 cells were analyzed on SFORM PC (VAMSTEC, Zagreb). Correlation between morphometric, AgNOR and ICM characteristics revealed the cells with low proliferative activity to possess small, homogeneous AgNOR, with the majority of cells in the peak of DNA histogram. The cells with high proliferative activity had inhomogeneous AgNOR, mostly containing greater DNA content than peak cells, pathologic mitoses (DNA > 4N), or the majority of cells were in the S-phase of the cell cycle. Cells with medium proliferative activity and annular AgNOR were in-between. Analysis of different tumor mass compartments showed that lymphatic cells with the affinity to accumulate in BM regularly exhibited low proliferative activity (a lower percentage of cells in SFC and highest percentage of cells in the peak of the G0/G1 phase). The cells in LN exhibited the characteristics of proliferative cells (an increased number of AgNOR, larger and more proliferative inhomogeneous AgNOR, and lowest percentage of cells in the G0/G1 phase). The migration of cells from BM to LN and between lymph nodes occurred through PB (there were cells with low and high proliferative activity: a higher proportion of cells in SFC and at the same time in the G0/G1 phase of the cell cycle). Analysis of cell size and proliferative activity in different compartments of tumor mass revealed that the cells in BM and PB did not differ substantially according to size and proliferative activity, while an inverse pattern was observed between PB and LN. As small cells are inactive and larger cells more proliferative, the analysis quite unexpectedly showed the PB cells to be largest and most inactive, in contrast to LN where the cells were smallest and most active. The "single" and "multiple programmed stops" have been hypothesized in the development of typical forms of leukemias and lymphomas and atypical forms of subacute and subchronic leukemias. Differentiation impairment may occur at any stage, and different "stop" locations result in different morphology and affinity to accumulation in bone marrow, peripheral blood and lymph nodes.