Characterisation of TP53 abnormalities in chronic lymphocytic leukaemia.

Abnormalities of TP53 in chronic lymphocytic leukaemia (CLL) correlate with aggressive disease and transformation. We studied 115 patients with CLL including 90 untreated, 25 with heavily pretreated/refractory CLL using fluorescent in situ hybridisation (FISH) to detect allelic loss at chromosome 17p and flow cytometry (FC) to test p53 protein overexpression. A total of 17 cases were identified with TP53 deletion and/or protein expression. Both tests correlated in 10 of 17 patients; in six, one or the other abnormality was detected and in one case, with a deletion, flow cytometry failed. Material for direct DNA sequencing was available in 14 of 17 cases. Mutations were found in seven cases. Five of 14 patients with allelic loss and seven of 13 expressing p53 protein had a mutation. These were single-base substitutions and were located in exons 5, 7 or 8. Mutations were not found in 13 of 14 other cases without deletions by FISH or protein expression. The incidence of p53 abnormalities in this series was 15%, with a significant difference between untreated patients (7%) and the pretreated/refractory group (50%; P<0.01). Abnormal p53 was predicted for shorter survival, regardless of the method used. We confirm that p53 abnormalities are more common in refractory CLL and that mutations occur at the known hot spots. Testing for TP53 deletions by FISH and protein expression by FC is an effective and simple way of screening patients who are likely to have aggressive disease. DNA sequencing adds little to these methods in identifying the population at risk.

Diagnostic significance of CD20 and FMC7 expression in B-cell disorders.

We analyzed by flow cytometry the expression of CD20 and FMC7 in cell suspensions from 932 patients, including 630 cases of chronic lymphocytic leukemia (CLL), 23 cases of other B-cell leukemias, and 279 cases of B-cell non-Hodgkin lymphoma (B-cell NHL). CD20 was positive in 94.5% of cases; FMC7 was positive in 35.7%. There was a correlation between CD20 and FMC7 expression in patients with B-cell NHL (P < .001) but not CLL (P = .1). We also tested a scoring system in which FMC7 was replaced by CD20 and compared it with our current scoring system for CLL. With this modification, the accuracy of the scoring system for differentiating CLL from other non-CLL disorders fell from 94.4% to 81.5%. In CD20+ CLL, the intensity of CD20 expression correlated with FMC7 and low scores (P < .001 for both comparisons). We suggest that the particular conformation of CD20 recognized by FMC7 is manifested only in cells with strong CD20 expression, which is not the case for CLL. FMC7 is of greater diagnostic value than CD20 for distinguishing CLL from other B-cell disorders; we recommend its continued use for this purpose.

High-affinity binding sites for heparin generated on leukocytes during apoptosis arise from nuclear structures segregated during cell death.

During cell death of human cultured leukocytes (Jurkat, HL-60, THP-1, U937) and freshly prepared leukocytes, we observed a greater than 100-fold increase in the affinity of apoptotic and necrotic cells for fluorescein isothiocyanate (FITC)-heparin in comparison with live cells. Binding of FITC-heparin was reversed in the presence of high ionic strength, unlabeled heparan sulfate, and heparin and pentosan polysulfate, but not in the presence of chondroitin and dermatan sulfates. During the course of cell death, the increase in the percentage of cells positive for annexin V binding correlated with the increase in the population positive for binding FITC-heparin. Confocal microscopy demonstrated that heparin binding to dead cells was restricted to 1 or 2 small domains on the surfaces of apoptotic cells and to larger, but still discrete, areas that did not localize with chromatin on ruptured necrotic cells. The heparin-binding domains originated from the nucleus and may correspond to the ribonucleoprotein-containing structures that have recently been shown to segregate within the nucleus of cells and to move onto the cell membrane. We observed that phagocytosis of dead Jurkat cells by monocyte-derived macrophages was blocked when the heparin-binding capacity of the dead cells was saturated by the addition of pentosan polysulfate. From this we concluded that the ability of dead cells to bind to heparan sulfate proteoglycans on the surfaces of macrophages may assist in phagocytic clearance.