Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system.

A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells x 10(6)/kg and CFU-C x 10(4)/kg was performed in 74 consecutive patients. The interval or 'gap' from the end of previous chemotherapy to the date of priming was recorded in weeks. Geometric mean CD34 and CFU-C values were significantly higher in patients with a score of < or = 60 compared to those with score > 60 (P = 0.003 and 0.02, respectively) and a significant difference in CD34 values was also found when scores of < or = 38 were compared with scores > 38 (P = 0.003), with the difference in CFU-C values approaching significance (P = 0.08). Patients exposed to toxicity factor 4 drugs had significant lowering of both CD34 and CFU-C values (P < 0.001 and P = 0.038) and this emerged as the only independent factor when analysed using linear regression. No significant difference in the geometric mean CD34 or CFU-C values of patients was found in any of the gap categories analysed. Prior exposure to toxicity factor 4 drugs had a significant adverse effect on stem cell yield and should be avoided or minimized prior to stem cell harvesting.

Bone marrow necrosis.

Bone marrow necrosis is most frequently diagnosed at postmortem examination. Antemortem diagnosis is still uncommon. In a recent review of world literature, we have found 133 cases of bone marrow necrosis diagnosed during life. It has been observed during the course of a wide variety of diseases, most commonly in association with acute and chronic leukemia, carcinoma, malignant lymphoma, infections, and sickle cell disease. We report two intravitally diagnosed cases of bone marrow necrosis occurring in two patients with high-grade B-cell lymphoproliferative disease. These cases are unusual in that both patients had a triad of bone marrow necrosis, high-grade B-cell lymphoproliferative disease, and hypercalcemia. Despite chemotherapy, both cases ultimately proved fatal, with progressive involvement of the central nervous system.

Inhibition of fibrin monomer polymerisation by myeloma immunoglobulin.

Myelomatosis was diagnosed in a 64 year old man on the basis of a serum paraprotein band (type IgG lambda, 42 g/l), plasma cell infiltration of bone marrow, and multiple lytic lesions evident on skull x ray picture. Blood specimens taken into plain glass tubes showed bulky gelatinous clot formation with minimal clot retraction. Coagulation tests were significantly abnormal with an increase in thrombin time, prothrombin time, and reptilase time. The possibility that the paraprotein was interfering with fibrin production was investigated. The rate of fibrin monomer polymerisation (measured turbidometrically) was reduced in patient plasma compared with control plasma. Although purified fibrin monomer prepared from the patient's fibrinogen polymerised normally, the addition of purified paraprotein caused a dose dependent reduction in the rate of polymerisation. These results suggest that the paraprotein was impairing fibrin formation by inhibiting fibrin monomer polymerisation. After chemotherapy the paraprotein concentration decreased and the coagulation results returned to normal.

Increase in mean platelet volume in patients with chronic renal failure treated with erythropoietin.

AIMS: To assess whether r-HuEPO (recombinant human erythropoietin) has any effect on thrombopoiesis in patients with chronic renal failure. METHODS: This was a retrospective study of 78 patients with chronic renal failure undergoing either haemodialysis (n = 57) or intraperitoneal dialysis (n = 21). All patients had a full blood count (in EDTA) measured before starting r-HuEPO and at monthly intervals thereafter up to six months. Variables studied were haematocrit, platelet count, mean platelet volume (MPV) and platelet distribution width (PDW). Other groups of control patients were also studied--patients with chronic renal failure receiving dialysis but not r-HuEPO (n = 40) and a group of patients with normal renal function who were receiving aspirin (n = 30). RESULTS: There was a significant increase in mean haematocrit (p < 0.01) and in mean platelet volume (p < 0.001) over the six month period, but no change in either total platelet count or platelet distribution width in the patients with chronic renal failure receiving r-HuEPO. In contrast, both the control groups showed no significant change in MPV. CONCLUSIONS: The results suggest that r-HuEPO affects thrombopoiesis and may be part of a group of humoral factors contributing to megakaryocyte development and maturation. Larger platelets are more reactive and may contribute to the increased risk of thrombosis associated with r-HuEPO.

B-cell chronic lymphocytic leukaemia populations respond stochastically to combinations of growth signals in vitro.

The ability of B-CLL lymphocyte populations to respond to cytokine stimulation of mononuclear cultures was examined using 3-colour immunofluorescence and multiparameter flow cytometry. The simultaneous detection of the membrane antigens CD5 and CD19, with the proliferation antigen Ki-67, allowed examination of the cell-cycle entry of the B-CLL lymphocyte population. Upon stimulation with IFN-gamma, IFN-gamma+IL-2, TPA+IL-4, between 10 and 44% of B-CLL lymphocytes from 10 patients were stimulated to enter into cell-cycle. This B-CLL response was notably more homogenous than the non-clonal lymphocyte response. Except in the case of IFN-gamma alone, T-cell response did not correlate with B-CLL response, suggesting B-CLL lymphocytes respond directly to combinations of growth signals. Considering the B-CLL population as homogenous, B-CLL lymphocytes respond in a stochastic manner.

Myeloma--results of treatment 1986-1990.

Sixty-nine patients with multiple myeloma diagnosed during a five year period at the Belfast City Hospital were followed until death or for a minimum of one year in a retrospective study of survival. Although the patients were unselected, survival data was found to be similar to results from trials in which patient selection had occurred. Overall median survival was thirty-two months. Median survival fell with advancing disease and was 47, 27 and 18 months for Durie-Salmon stages I, II and III respectively. Those patients presenting with a platelet count of < 100 x 10(9)/1 had a median survival of eight months in contrast to those with a platelet count > 100 x 10(9)/1 whose median survival was 36 months. Patients presenting in renal failure had a shorter median survival of 28 months compared to 46 months for those with normal renal function.

Normal unstimulated lymphocytes produce granulopoietic inhibitory activity.

Normal unstimulated lymphocytes cocultured with normal bone marrow will inhibit day-7 colony- forming units in culture. We have shown that this phenomenon has a molecular basis and attempted to characterize it further. Using specific assays and neutralizing monoclonal antibodies we have found that this granulopoietic inhibitory activity (GIA) is not due to alpha- or gamma-interferon, tumor necrosis factor, or acidic or basic isoferritins. Biochemical studies suggest that it is a glycoprotein with a molecular weight greater than 100,000 daltons. It appears to act on cells in S phase, although it may not be S-phase specific. GIA represents a novel inhibitor that merits further investigation.

The proliferative activity of B-chronic lymphocytic leukaemia lymphocytes prior to and after stimulation with TPA and PHA.

The proliferative activity of B-CLL lymphocytes from 10 patients was investigated both prior to and after stimulation with TPA and PHA. The analysis of cell cycle-associated features such as BrdU incorporation and the expression of the nuclear proliferation-associated antigen, Ki-67, together with the phenotypic profile of the cells, was performed using double colour immunofluorescent methods. The unstimulated B-CLL cells represented a homogeneous population with the same cell cycle position (G0) as resting peripheral blood lymphocytes. After TPA stimulation 22.7% of the lymphocytes were found in G1, 9.4% in S + G2/M and 13.4% in post-M. PHA stimulation induced a greater proportion of cells in G1, i.e. 35% and 17.8% into S + G2/M and 13.4% into post-M. Double colour immunofluorescence was able to demonstrate that in TPA cultures the majority of the stimulated lymphocytes originated from the malignant clone. Evidence of B-CLL lymphocyte proliferation using double colour labelling with BrdU and Ig kappa and/or Ig lambda showed that a small minority of B-CLL lymphocytes were stimulated into S + G2/M phases of the cell cycle. PHA was also capable of inducing a small proportion of B-CLL cells into mitosis although this proportion of cells was smaller compared to the TPA-stimulated lymphocytes.

An Analysis of Circulating CD4 Lymphocyte Subpopulations in B-Cell Chronic Lymphocytic Leukaemia.

The distribution of circulating CD4 lymphocyte subpopulations determined by reactivity with monoclonal antibodies anti-2H4 (CD45RA), anti-UCHL1 (CD45RO), anti-4B4 (CD29) and anti-Leu8, and analysed by dual colour immunofluorescence flow cytometry is described in a series of patients with B-CLL and in age-matched control subjects. The percentages and absolute numbers of CD4 cells reactive with anti-CD45RA, anti-CD45RO and anti-CD29 reagents were similar in the patient and control groups. In contrast, CD4+ Leu8+ cells (percentages and absolute numbers) were significantly reduced in B-CLL patients resulting in an inversion of the normal CD4+ Leu8+ :CD4+Leu8- ratio. The patients' clinical or therapeutic status did not appear to influence the levels of the respective CD4 subpopulations; nor was evidence of hypogammaglobulinaemia associated with specific numerical alterations in any of the CD4 subpopulations studied. It is proposed that, in B-CLL, the alterations in the CD4Leu8 subpopulations are associated with the disease process, whereas the distributions of CD4+CD45RA+, CD4+ CD45RO+ and CD4+ CD29+ cells reflect the normal physiological levels of these subpopulations in elderly subjects.

Lymphocytes from patients receiving lithium do not inhibit CFU-C growth.

Lymphocyte subset levels and function were examined in 12 patients on lithium therapy and in 11 healthy hospital personnel. Co-culture of allogeneic human bone marrow cells with monocyte-depleted lymphocyte preparations revealed that CFU-C formation was significantly reduced (mean 43% inhibition) in the presence of normal lymphocytes but not with the patients' lymphocytes (less than 5% inhibition). This did not reflect numerical changes in lymphocyte subsets, since these were similar for control and lithium subjects. T colony formation was significantly depressed in the patient group (P less than 0.05), whereas B colony numbers were similar in both groups (P greater than 0.1). The possible role of HLA-incompatibility affecting CFU-C growth was investigated in co-culture experiments, using lymphocytes from HLA-identical twins, one of whom was receiving lithium. In four separate co-culture experiments, the inhibitory effect was shown with lymphocytes from the non-lithium twin but was not demonstrated by the lithium subject. Addition of lithium in vitro to co-cultures of normal marrow and lymphocytes was found to negate the inhibitory phenomenon in a dose-related manner. It is postulated that granulocytosis induced by the administration of lithium may be a manifestation of changes in a lymphocytic control system.

Renal function in the elimination of oral melphalan in patients with multiple myeloma.

Pharmacokinetic studies in 11 patients with multiple myeloma were undertaken on the first and last days of one course of chemotherapy. The drug was administered PO in single doses of 6-14 mg daily. Melphalan concentrations were determined by high-performance liquid chromatography. The interpatient variability of pharmacokinetic parameters noted by other authors was observed. Regression analysis showed a significant positive correlation between the elimination rate constant for melphalan and renal function (P = 0.003). The form of the line which describes the overall elimination rate constant for melphalan is given by the equation: Kel = 5.67 X 10(-3) + [4.90 X 10(-5) X GFR]. There was also a significant negative correlation between renal function and the area under the plasma melphalan concentration/time curve (P = 0.006). In vitro stability studies of melphalan in plasma at 37 degrees C and pharmacokinetic data suggest that hydrolysis and renal clearance are the major mechanisms of melphalan elimination. This work shows quantitatively the relationship between renal function and drug elimination and how the data may be used in predicting melphalan half-life from creatinine clearance.

Can food affect the bioavailability of chlorambucil in patients with haematological malignancies?

Pharmacokinetic studies in ten patients with haematological disorders were undertaken on the first and second days of one course of chemotherapy. Patients received chlorambucil under fasting and non-fasting conditions. Plasma concentrations of chlorambucil were determined by a reversed-phase high-performance liquid chromatography assay. Statistical analysis by the Wilcoxon signed rank test for non-parametric data indicated that food caused a significant reduction in peak plasma levels (P less than 0.01), elimination rate constants (P less than 0.01) and area under the plasma chlorambucil/time curve (P = 0.01). Food was also found to prolong the time taken to attain peak plasma levels (P less than 0.01). Regression analysis of renal function with elimination rate constants showed that chlorambucil elimination was independent of renal function (n = 8; r = -0.007; P = 0.72). In view of these results we suggest that chlorambucil is given on an empty stomach.

Circulating T-cell subpopulations in lithium-associated granulocytosis.

Granulocytosis is a common feature in patients undergoing lithium therapy. With increasing evidence that T lymphocytes play a role in the control of granulopoiesis, we have investigated the effect of lithium administration on circulating levels of T helper and T suppressor cells, as identified by monoclonal antibodies, to determine whether lithium-induced granulocytosis is mediated through changes in peripheral blood T cell subsets. Lithium carbonate was administered to 10 subjects over a 2 week period. Differential leucocyte counts and T, B, T helper and T suppressor lymphocyte enumerations were performed prior to administration of lithium (Day 1) and on 2 occasions (Day 7 and 14) during ingestion of the drug. Ten healthy control subjects were similarly investigated. Small, but significant elevation (p less than 0.05) in neutrophil counts at 7 and 14 days were observed in subjects taking lithium, serum lithium levels at these times were 0.56 +/- 0.27 and 0.68 +/- 0.17 mmol/l, respectively; lymphocyte and monocyte levels were unaffected. The percentages and absolute numbers of circulating T, B, T helper and T suppressor lymphocytes were not significantly altered (p greater than 0.05) during lithium administration and did not differ significantly (p greater than 0.05) from those recorded for the control group. We were thus unable to demonstrate that short-term lithium administration induced changes in the circulating levels of T helper (OKT4+) or T suppressor (OKT8+) cells.

Can severe vincristine neurotoxicity be prevented?

Neurotoxicity in vincristine treatment has generally been considered a consequence of the cumulative dose of the drug, and liver dysfunction had been recognised as an indication to reduce the dosage. We demonstrate that neurotoxicity is also related to individual doses and that even when there is no other evidence of liver dysfunction, a raised level of serum alkaline phosphatase may predict severe neurotoxicity. Exposure to vincristine following IV injection of the drug was studied in 27 subjects by measuring the area under the vincristine plasma concentration time curve (AUC 0-infinity). A statistically significant relationship was found between the AUC0-infinity and the degree of neurotoxicity. The AUC0-infinity was related both to dose and to elevation of serum alkaline phosphatase, suggesting that elimination of the drug is impaired when serum alkaline phosphatase is raised. Among patients with elevated serum alkaline phosphatase, a small reduction in the dose of the drug resulted in lower vincristine plasma AUC0-infinity and less neurotoxicity.