Diagnostic accuracy of extended biopsies for the staging of microfocal prostate cancers in autopsy specimen.

Clinically insignificant prostate cancers may be predicted when biopsies show a microfocal cancer (MiFC). However, at least one-third of MiFC are underestimated by biopsies. The aim of this study was to evaluate the staging accuracy of different biopsy regimen showing a MiFC. We performed 18 biopsy cores on 164 autopsy prostates. Six cores were taken from the mid-peripheral zone (MPZ), 6 from the lateral PZ (LPZ) and 6 from the central zone (CZ). We tested seven different biopsy regimens by distinguishing the MPZ, LPZ or CZ biopsies either separately or associated with each other. Of the cancers detected by biopsies, we selected those showing a MiFC and compared our findings with whole mount analysis. The positive predictive value of a MiFC referred to how often, when needle biopsies showed a MiFC, there was a clinically insignificant cancer on whole mount prostate analysis. We found that the positive predictive value of a MiFC on 6 or 12 biopsy cores was similar irrespective of biopsy location (P approximately 1). On MPZ, MPZ plus LPZ and all 18 biopsies, it was 40, 70 and 87%, respectively (P<0.1). Tumor volume of cancers showing a MiFC on MPZ biopsies was significantly higher than those showing a MiFC on MPZ plus LPZ, or all 18 biopsies (P<0.05). These results show that performing additional cores in case of MiFC on sextant biopsies may help differentiating significant from insignificant cancers.

Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia.

BACKGROUND: Fludarabine is an effective treatment for chronic lymphocytic leukemia that does not respond to initial treatment with chlorambucil. We compared the efficacy of fludarabine with that of chlorambucil in the primary treatment of chronic lymphocytic leukemia. METHODS: Between 1990 and 1994, we randomly assigned 509 previously untreated patients with chronic lymphocytic leukemia to one of the following treatments: fludarabine (25 mg per square meter of body-surface area, administered intravenously daily for 5 days every 28 days), chlorambucil (40 mg per square meter, given orally every 28 days), or fludarabine (20 mg per square meter per day for 5 days every 28 days) plus chlorambucil (20 mg per square meter every 28 days). Patients with an additional response at each monthly evaluation continued to receive the assigned treatment for a maximum of 12 cycles. RESULTS: Assignment of patients to the fludarabine-plus-chlorambucil group was stopped when a planned interim analysis revealed excessive toxicity and a response rate that was not better than the rate with fludarabine alone. Among the other two groups, the response rate was significantly higher for fludarabine alone than for chlorambucil alone. Among 170 patients treated with fludarabine, 20 percent had a complete remission, and 43 percent had a partial remission. The corresponding values for 181 patients treated with chlorambucil were 4 percent and 33 percent (P< 0.001 for both comparisons). The median duration of remission and the median progression-free survival in the fludarabine group were 25 months and 20 months, respectively, whereas both values were 14 months in the chlorambucil group (P<0.001 for both comparisons). The median overall survival among patients treated with fludarabine was 66 months, which was not significantly different from the overall survival in the other two groups (56 months with chlorambucil and 55 months with combined treatment). Severe infections and neutropenia were more frequent with fludarabine than with chlorambucil (P=0.08), although, overall, toxic effects were tolerable with the two single-drug regimens. CONCLUSIONS: When used as the initial treatment for chronic lymphocytic leukemia, fludarabine yields higher response rates and a longer duration of remission and progression-free survival than chlorambucil.

Usefulness of the mean platelet volume.

The mean platelet volume measurement can reflect changes in either the level of platelet stimulation or rate of platelet production. The controversy related to whether platelets change volume or density in the circulation is still not clearly resolved, and although the relationship between platelet number and size and megakaryocyte number, size, and ploidy have been well described, the factors that regulate this interaction are still poorly understood. Platelet volumes are frequently measured after being collected in EDTA and stored at room temperature in spite of the known artifacts that are induced by this type of specimen handling. Still, the MPV can be useful in selected clinical conditions. If platelet consumption in vascular disorders is associated with stimulated platelet production, however, then there is a need for further optimization of this technique. Proper method standardization and the development of more stable specimen handling conditions may allow the MPV to have broader clinical application in detecting and monitoring vascular disorders. Development of improved technique may currently be impeded by the ease with which highly artifactual measures can be now be obtained.

Immunophenotypic subclassification of chronic lymphocytic leukaemia (CLL).

To determine the significance of the immunophenotypic heterogeneity of B-cell chronic lymphocytic leukaemia (CLL), surface immunoglobulins (SIgs), mouse rosette assays (MR), and a panel of monoclonal antibodies for B cells, T cells and myeloid cells were performed on peripheral blood samples from 61 newly diagnosed cases. Four groups were observed: group I (SIg+, MR+, CD19/20+, CD5+, T antigen (Ag)-; 27 cases); group II (SIg+, MR+, CD19/20+, CD5+, T Ag+; 17 cases); group III (SIg+, MR+ CD19/20+, CD5-, T AG-; 12 cases); and group IV (SIg-, MR+, CD19/20+, Cd5+, T Ag-; 5 cases). Groups were compared according to French-American-British Cooperative Group subtypes, clinical and laboratory features, Rai staging, and survival. Typical CLL morphology (greater than 90% small lymphocytes) was present in 20/20 (100%) of group I cases and 23/27 (85%) group II, III and IV cases (P = 0.09). Expression of a myeloid antigen was seen in 5/27 group I cases (18%) and 1/16 group II cases (6%), but was not predictive of survival (P = 0.36). The CD5- group III had a lower haemoglobin level (P less than 0.0001), higher Rai stage (P less than 0.002), and poorer survival at 5 years (P less than 0.02) than the other groups. We conclude that at least four distinct immunophenotypic subgroups of B-cell CLL can be determined. Expression of myeloid or T-cell antigens does not appear to predict for patient survival; however, lack of CD5 antigen may be associated with more advanced stage of disease and poor patient survival.

Temperature-dependent binding of cyclosporine to an erythrocyte protein.

In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [3H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA.

Evidence of a cyclosporine-binding protein in human erythrocytes.

To investigate the phenomenon of different erythrocyte saturation capacities for cyclosporine (CsA) in the blood of different individuals, hemolysates of washed red cells were examined for the presence of a CsA-binding protein. Using gel filtration column chromatography of hemolysates from patients receiving CsA orally, the majority of erythrocyte-associated CsA eluted as a single peak with Mr 15,000-17,000, distinct from hemoglobin and carbonic anhydrase. [3H]CsA added to a hemolysate in vitro eluted similarly. [125I]CsA added to a hemolysate eluted much later in the same position as [3H]CsA mixed with albumin and myoglobin (presumably as free unbound drug). These findings indicate that CsA normally binds to an intraerythrocytic protein similar in molecular size to calf thymus cyclophilin (Mr 15,000). By equilibrium dialysis, the purified erythrocyte proteins calmodulin (Mr 16,700) and cytochrome b5 (Mr 15,000) failed to bind CsA. By equilibrium dialysis, [3H] CsA did bind to column fractions containing the CsA-binding protein, but [125I]CsA did not, suggesting that attachment to CsA occurs at or near a carbon-carbon double bond in an unusual nine-carbon amino acid of CsA. These results have important implications for CsA therapy with regard to distribution space, pharmacokinetics, and a possible protein-receptor mechanism of action.

Hydroxyurea interferes negatively with triglyceride measurement by a glycerol oxidase method.

Measured triglyceride concentrations were extremely low (less than 100 mg/L) in the serum of some patients who were receiving hydroxyurea for myeloproliferative diseases. The assay being used to quantify triglycerides was a "cascaded" enzymatic method involving (a) lipase, to generate glycerol from triglycerides; (b) glycerol oxidase, to convert glycerol to glyceraldehyde, with generation of hydrogen peroxide; and (c) peroxidase, which acts on the hydrogen peroxide with subsequent coupled generation of a red-violet quinone (reagent system used in the Technicon RA-1000). Hydroxyurea added to serum samples appeared to inhibit the action of glycerol oxidase, with a stoichiometric relation to the concentration of substrate (a decrease of roughly 2.4 mmol/L in measured triglyceride per 1 mmol of hydroxyurea per liter). A different enzymatic assay for triglycerides, which involves glycerol kinase (Beckman Instruments) did not show this effect of hydroxyurea.

Specificity of autoantibodies to lactate dehydrogenase isoenzyme subunits.

We studied serum from 12 unrelated patients with variant electrophoretic patterns for lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes characteristic of LD-immunoglobulin complexes. Immunoglobulin class was identified in four cases. In all 12 cases, mixing the patient's serum with control serum altered the mobility of normal LD isoenzymes to that of the variant. To determine antigenic specificity, we mixed ammonium sulfate-precipitated fractions of patient's samples with purified LD1 (H tetramer) or LD5 (M tetramer) prepared by anion-exchange chromatography. In six cases the fractionated antibody displayed a pure M-subunit specificity. In four cases it acted against both M tetramers and H tetramers, and in two cases the antibody affected the migration only of LD isoenzymes containing both M and H subunits (i.e., hybrid isoenzymes). Depending on the relative excess of antibody or LD5 in such mixtures, various different anomalous electrophoretic patterns were produced. These results indicate that apparent differences between variant LD isoenzyme patterns in whole serum of different individuals can arise from autoantibodies with similar reactivities being present in various ratios of antigen to antibody.

Assessment of cyclosporin A in whole blood and plasma in five patients with different hematocrits.

Measurement of whole blood and plasma levels of cyclosporin A (CsA) by radioimmunoassay in kidney transplant recipients receiving the drug showed that CsA concentrations in plasma increased nonlinearly when whole blood levels of the drug exceeded 1,000 ng/ml. At low plasma levels (less than 200 ng/ml), most of the CsA in the blood was in the nonplasma component, indicating that cellular elements have high affinity for CsA. Comparison of nonplasma CsA concentrations in two patients with hematocrit values of 32.5 and 35% showed that in the patient with a hematocrit of 35% the cellular associated drug was twice as great as that in the other patient, indicating that there may be significant differences in the cellular affinity of CsA in patients with similar hematocrits. Linear regression analysis of the cellular associated CsA versus plasma levels of the drug in a double-reciprocal plot showed a drug saturation capacity of 6,060 ng/ml in the nonplasma component of blood in the patient with a hematocrit of 35%. Similar analyses in the other patients indicated saturation capacities ranging from 4,750 to 10,400 ng/ml.

Modulation of radiation-induced hemopoietic suppression by acute thrombocytopenia.

Modifications of radiation-induced hemopoietic suppression by acute thrombocytopenia were evaluated. Immediately before or after exposure to sublethal irradiation, mice were given a single injection of anti-mouse platelet serum (APS), normal heterologous serum, neuraminidase (N'ase), or saline, or no further treatment was provided. Hemopoiesis was evaluated by blood cell counts, hematocrits, and incorporation of [75Se]selenomethionine into platelets. APS and N'ase induced an acute thrombocytopenia from which there was partial recovery before the platelet count started to fall from the radiation. During the second post-treatment week, both thrombocytopoiesis and erythropoiesis were greater in mice that received APS or N'ase in addition to radiation than in control irradiated mice. Differences in leukopoiesis were not apparent. Therefore, both thrombocytopoiesis and erythropoiesis appeared to be responsive to a stimulus generated by acute thrombocytopenia in sublethally irradiated mice.

Mean platelet volume: the need for a reference method.

The time course of artifactual effects due to anticoagulants, specimen temperature, and interval between venipuncture and analysis on platelet volume measurements was evaluated. Split specimens were analyzed using hydrodynamic focusing, and platelet distributions were computed using a least-squares fit to a log-normal distribution. Significant artifacts resulted from exposure to EDTA, cooling to room temperature, and delay in exposure to anticoagulant. The artifactual effect of EDTA is extreme and time dependent. Collection of blood in Buffered Citrate, Acid Citrate Dextrose, or Pyridoxal-5'-phosphate supplemented Citrate yielded stable and equivalent results with rapid anticoagulation and incubation at 37 degrees C for up to six hours.

Characterization of apparent lactate dehydrogenase isoenzyme 6: a lactate-independent dehydrogenase.

We found in a prospective study of lactate dehydrogenase isoenzyme 6 (LD6) in human tissues obtained at autopsy that LD6 was usually present in liver when death was preceded by prolonged hypotension or impaired ventilation but not in cases of sudden death. Other organs containing LD6 were kidney and spleen. LD6 is heat stable, differs from H-subunit-containing LD isoenzymes by pyruvate resistance, differs from M-subunit-containing LD isoenzymes by immunoprecipitation, and is distinct from spermatic LDX. LD6 from liver extracts acted without lactate as substrate and could be enhanced by ethanol added to the substrate. These results indicate that LD6 is not a true lactate dehydrogenase, and that it frequently appears in severe liver injury.

Megakaryocytopoiesis in irradiated, splenectomized mice.

The hypomegakaryocytic state that develops after exposure to sublethal doses of ionizing radiation was evaluated in splenectomized and intact mice. The percentage reduction of marrow megakaryocytes was greater than that of platelets at comparable times post-irradiation. After initial recovery a secondary drop in platelet counts occurred earlier in intact than in splenectomized mice. The average size of mature megakaryocytes was found to be increased, due primarily to marked reductions in megakaryocytes of smaller size. These results indicate that the spleen acts more to reduce than to increase the platelet count after exposure to sublethal doses of whole body radiation and that megakaryocyte size may be increased by reduction in numbers of small megakaryocytes without an increase in large megakaryocytes.