Can flow cytometry of bone marrow aspirate predict outcome of patients with diffuse large B cell lymphoma? A retrospective single centre study.

Bone marrow (BM) trephine biopsy is a part of routine staging of patients with newly diagnosed diffuse large B cell lymphoma (DLBCL). The significance of lymphoid monoclonal population on flow cytometry (FC) of the BM aspirate in the presence of negative BM histology has not been clarified. In this study, we assessed the clinical role of positive FC in predicting outcome of patients with DLBCL and a negative BM histology. We retrospectively analysed 101 patients diagnosed with DLBCL at a single institution between years 1994-2003. Three groups of patients were compared: patients with histologic involvement of the BM (BM+), patients with no histologic involvement of the BM but with positive FC (BM-FC+) and patients with neither histologic or FC evidence of BM involvement (BM-FC-). The BM+ group included 13 patients (13%). The BM-FC+ group 16 patients (16%), and the BM-FC-included 72 patients (71%). Median age of the cohort was 67 years. Disease stage and International Prognostic Index score were significantly higher in the BM+ and BM-FC+ groups compared with the BM-FC- group. Median overall survival (OS) for the BM-FC-, BM-FC+ and BM + groups were 4.6, 2.2 and 0.9 years, respectively. Median progression free survival (PFS) for the BM-FC-, BM-FC+ and BM+ groups were 3.2, 1.4 and 0.6 years, respectively (p=0.01 for both analysis). In multivariable Cox regression models adjusting for age, sex, stage and International Prognostic Index, there was no significant differences in OS or PFS between the BM-FC+ and BM-FC- groups. In conclusion, positive FC in the setting of negative BM histology at diagnosis did not significantly affect OS or PFS.

Overexpression of tetraspanins affects multiple myeloma cell survival and invasive potential.

Cellular interactions with microenvironmental components are critical in multiple myeloma (MM) and impede effective disease treatment. Membranal-embedded tetraspanins, associated with metastasis suppression, are underexpressed in MM. We aimed to investigate the consequences of CD81/CD82 tetraspanins over-expression in MM cell lines. CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81/CD82. Employing flow cytometry, immunocytochemistry, and activity assays we assessed transfected cells for: morphology, survival, death, caspases, cell cycle, proliferation, oxidative stress, adhesion, motility and invasion. Overexpressed CD81/CD82 pEGFP-N1 vectors reduced survival without elevation of pre-G1 or AnnexinV+/7AAD- and independently of caspases. Decreased Ki67 and elevated intracellular glutathione were detected. No perturbations in cell cycle distribution were observed. The pEGFP-C1 vectors of CD81/CD82 caused reduction of MM cell adherence with/without fibronectin, insulin-like growth factor (IGF)-I, and matrigel. They also reduced cell motility and attenuated invasion potential, expressed by reduced secreted MMP-9 activity. These novel findings delineate the significance of CD81/CD82 expression to MM cell survival and their negative effects on cell adhesion, motility, and invasion thus, supporting their role as tumor metastasis suppressors.

Should bone marrow examination be routinely performed for the diagnosis of monoclonal gammopathy of undetermined significance?

BACKGROUND: Monoclonal gammopathy of undetermined significance is defined by the presence of: low serum and/or urine monoclonal protein level; less than 10% plasma cells in bone marrow; normal serum calcium, creatinine and hemoglobinlevels; and no bone lesions on full skeletal X-ray survey. OBJECTIVES: To study the necessity of bone marrow examination for the diagnosis and clinical course of MGUS. METHODS: We retrospectively screened the medical records of all patients in whom monoclonal protein was found in the serum during 2001-2002 in the medical laboratories of Meir Medical Center. Asymptomatic patients who had serum monoclonal immunoglobulin G < 3.0 g/dl or IgA < 2.0 g/dl or IgM < 1.0 g/dl without anemia, renal failure, hypercalcemia or any bone lesions on skeletal survey were eligible. Full records of patients who were evaluated in the hematology clinic were available (group 1). The remaining patients were followed by their family physicians; thus we had access only to their electronic files including laboratory results and new diagnoses (group 2). Demographic and clinical parameters as well as clinical course werewere evaluated. RESULTS: Both groups (57 and 255 patients, respectively) had similar demographic, laboratory and clinical characteristics. Bone marrow examination was performed in 30 of 57 patients (group 1): 16 were normal, 8 had an excess of normal plasma cells, and 6 had excess of pathologic plasma cells. However, only in two of the latter six could a diagnosis of multiple myeloma be established. All group 1 patients were followed for 22 +/- 11 months and onlytwo developed overt multiple myeloma. During the same period, 6 of 255 patients (group 2) were diagnosed as multiple myeloma and 3 as MGUS in other hospitals. The rest had a stable course with no change in their laboratory values. CONCLUSIONS: Our findings suggest that bone marrow examination should not be performed routinely in patients who fulfill strict clinical and laboratory criteria of MGUS.

Nonmyeloablative conditioning does not prevent telomere shortening after allogeneic stem cell transplantation.

BACKGROUND: Stem cell transplantation (SCT) may be associated with premature aging of the hematopoietic stem cells. Telomere length reflects the proliferative history of a cell. In most studies published so far on telomere dynamics after myeloablative allogeneic SCT, recipients had shorter telomeres than their respective donors, thus reflecting "accelerated aging" of hematopoietic cells. We evaluated telomere dynamics in patients who underwent transplantation with nonmyeloablative protocols, assuming that the decreased intensity of chemotherapy might prevent telomere attrition. METHODS: Telomere length was measured using FISH-FACS method. Telomeres of recipients were compared to their respective donors. Twenty-three consecutive patients after nonmyeloablative SCT were evaluated. A control group consisted of 10 donor-recipient pairs after conventional myeloablative transplantation. RESULTS: There was significant telomere shortening in both recipients of nonmyeloablative and myeloablative conditioning (0.487+/-0.65 kb, P=0.003; 0.361+/-0.50 kb, P=0.047 respectively). The extent of telomere shortening in the two groups was not different (P=0.64). There was no correlation between the degree of shortening and parameters such as time interval from transplant, age of donor or recipient, and the number of infused cells. CONCLUSIONS: This is the first study on telomere dynamics after nonmyeloablative conditioning SCT. The study demonstrates significant shortening of telomeres in recipients in spite of decreased intensity conditioning. Results of this study suggest that the main mechanism following transplantation is the proliferative stress imposed upon the stem cells and not direct damage by cytotoxic drugs. The different kinetics of restoration of hematopoiesis and the probable ongoing process of graft-versus-leukemia in the bone marrow do not prevent the attrition of telomeric ends of chromosomes.

Simvastatin induces apoptosis of B-CLL cells by activation of mitochondrial caspase 9.

BACKGROUND AND OBJECTIVES: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Despite several advances in therapeutic options, the disease remains incurable. Recently, it was repeatedly demonstrated that statins, competitive inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have antineoplastic effects. Therefore we aimed to study the effects of simvastatin (Sim) on malignant B cells derived from patients with CLL and mechanisms of action of the drug. METHODS AND RESULTS: Purified B-CLL cells from 15 patients were cultured either alone or with Sim at concentrations of 10, 50, and 100 microM. Viability, measured by the activity of mitochondrial dehydrogenases, was reduced significantly in the cells treated with Sim at 50 and 100 microM for 24 hours (p<0.005). The level of apoptosis, as measured by annexin binding to exposed phosphatidylserine moieties, increased significantly in the treated cells at concentrations higher than 50 microM for 24 hours (p<0.003). The level of necrosis, as measured by propidium iodide internalization, increased significantly after 24 hours exposure to Sim at 50 microM (p<0.01). The apoptotic cascade was studied by immunoblot analysis of caspases following Sim treatment. These showed cleavage of caspases 9, 8, and 3. Addition of the caspase inhibitor Z-VAD.fmk inhibited caspase 8 and 3 significantly but did not affect caspase 9. CONCLUSION: Exposure of clonal B lymphocytes from patients with CLL to simvastatin decreases viability significantly by the induction of apoptosis. The apoptosis induced by Sim is probably initiated by the mitochondrial caspase 9, which indirectly leads to activation of caspase 3 and 8.

Tamoxifen enhances apoptotic effect of cisplatin on primary endometrial cell cultures.

BACKGROUND: Cisplatin (CDDP) dose-limited by its side-effects is, in some instances, synergistically amplified when combined with tamoxifen (TAM). TAM has been shown to modulate apoptotic pathways of normal endometrial cells, whereas CDDP induces apoptosis in malignant endometrial cells. Their combined effect on normal or malignant endometrium is as yet unknown. This study aimed to evaluate the combined CDDP and TAM's apoptotic effect on normal endometrial tissue in the context of hormonal milieu. MATERIALS AND METHODS: Primary endometrial cell cultures were established and maintained both in the presence and absence of steroidal hormones. The cultures were treated for 24 hours with 20 microM TAM and 50 microM CDDP as single drugs and in combination. Apoptosis was determined by evaluation of pre G1 cell populations in the cell cycle analysis with flow cytometer. RESULTS AND CONCLUSION: CDDP induced apoptosis in all cultures regardless of hormonal environment, while TAM significantly enhanced CDDP-induced apoptosis in steroidal deficient media in an additive manner. These are novel findings depicting CDDP's effect on normal endometrium, singularly and combined with TAM.

Simvastatin induces death of multiple myeloma cell lines.

BACKGROUND: Accumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy. METHODS: U266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 microM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity. RESULTS: Exposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 microM for ARH77, 8 microM for RPMI 8226, and 13 microM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected. CONCLUSIONS: Simvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

Phosphatidylserine expression on the platelet membrane of patients with myeloproliferative disorders and its effect on platelet-dependent thrombin formation.

Recently, the asymmetric distribution of phospholipids in eukaryotic cell membranes has been appreciated and been found to be dependent on the activity of a number of enzymes. The expression of phosphatidylserine (PS), a negatively charged phospholipid, on the platelets of patients with polycythemia vera (P vera) and essential thrombocythemia (ET) was compared to that in normal individuals. The effect of platelet aggregation on PS expression was determined. Exposure of PS on platelets obtained from patients with P vera and ET and from age- and sex-matched healthy volunteers was measured by fluorescein-labeled Annexin V binding to platelets and by the platelets' thrombin-generating capacity determined by the prothrombinase assay. PLatelet prothrombinase activity (mean +/- standard deviation [SD]), as measured by thrombin generation, was 2.32+/-2.2 micro/mL in the P vera group and 1.55+/-1.0 micro/mL in the control group (p=0.3). PS expression as measured by Annexin V binding (mean +/- SD) was 2.6+/-2.4 % in the P vera group versus 1.55+/-1.2% among controls (p=0.03). In the ET group, prothrombinase activity (mean +/- SD) was 1.0+/-0.6 micro/mL and 2.1+/-0.9 micro/mL in the control group (p=0.006). Annexin V binding (mean +/- SD) was 4.8+/-4.2% in the ET group and 2.77+/-2.1% among control subjects (p=0.09). When the prothrombinase assay was performed after addition of adenosine diphosphate (ADP) to the platelets, there was a significant increase in thrombin generation in the myeloproliferative disorder (MPD) group (3.1+/-2.0 micro/mL) compared to the thrombin generated by unstimulated myeloproliferative disorder platelets (2.07+/-1.69 micro/mL) (p=0.0006). An increase in thrombin generation was seen in the ADP-stimulated platelet samples in all ten paired samples studied. Likewise, the addition of ADP to control platelets increased thrombin generation from 2.0+/-1.0 micro/mL in unstimulated platelets to 4.3+/-1.6 micro/mL in ADP-treated platelets (p=0.0006). Thrombin generation increased in all of the ADP-stimulated platelet samples compared to the untreated platelets. There was however, no difference in the increased thrombin generation when ADP-stimulated platelets from MPD patient and control subjects were compared (p=0.3). Results indicate that some patients with MPDs may show increased PS expression on platelet surface. When analyzed overall, there was a tendency toward greater PS expression in the P vera and ET patient groups; however, the increase did not reach statistical significance. This increase was noted in both the prothrombinase assay the Annexin V binding assay. We have also shown that stimulation of platelets by addition of the agonist ADP results in enhanced PS expression, which appears increase the thrombogenic potential of the platelets as demonstrated by the enhanced thrombin generation demonstrated by these platelets in the prothrombinase assay. There was no difference in the degree of PS expression in response to ADP stimulation between MPD and control platelets. Results show that PS expression and platelet-dependent thrombin generation is variable in patients with MPDs. This expression is increased after platelet aggregation occurs. The role of PS expression in the thromboembolic complications of MPD patients should be studied further.

The effect of plasma depletion of platelet concentrates on platelet aggregation and phosphatidylserine expression.

Plasma-reduced platelet concentrates are commonly administered to prevent febrile transfusion reactions and to avoid fluid overload in neonates. Because little is known about the influence of centrifugation and resuspension on functional aspects of platelets, we examined the effects of plasma-reduction on platelet aggregation and platelet-dependent thrombin generation. Our results show that plasma reduction and resuspension of the platelet pellet in saline or plasma results in a significant reduction in platelet aggregation to a combination of the platelet agonists adenosine diphosphate and epinephrine (p < 0.001). In contrast, when a combination of the more potent agonists collagen and thrombin was used, platelet aggregation was maintained. Likewise, no decline was observed in platelet-dependent thrombin generation as measured by the functional prothrombinase assay or Annexin V binding. We conclude that centrifugation and resuspension of platelets to render the concentrate plasma-free, as a routine procedure in blood banking, variably affects in vitro platelet aggregability but does not significantly affect platelet-dependent thrombin generation.

Promoter hypermethylation of tetraspanin members contributes to their silencing in myeloma cell lines.

Multiple myeloma (MM) cell interactions with their microenvironment modulate acquired drug resistance and disease progression. Indeed, reported aberrant gene methylation underscores the possible role of epigenetic events in MM's molecular profile. Membranal tetraspanins are often inversely correlated with cancer prognosis and metastasis, however mutations were unidentified hitherto. Their promoter characteristics and frequent down-regulation conform to transcriptional silencing by chromatin remodeling. We delineated the baseline expression of select tetraspanins in MM cell lines (RPMI 8226, U266, ARP1, ARK, CAG and EBV transformed ARH77) and fresh bone marrow samples (n = 9) for the first time and determined reduced expression of CD9, CD81 and absence of CD82. Thus, we aimed to assess their promoter methylation status. Indeed, we established CD9, CD81 and CD82 promoter methylation in MM cell lines employing methyl-specific-PCR of bisulfite modified G-DNA and PCR of G-DNA digested with methylation-sensitive restriction enzyme (Hin6I). Re-transcription of assayed genes in the cell lines following de-methylation [5-aza-2'-deoxycytidine (5-aza-dC)] confirmed the mechanistic significance of methylation to their regulation. Combined de-methylation and de-acetylation [Trichostatin A (TsA)] induced synergistic elevation of CD82 mRNA. We conclude that chromatin remodeling contributes to tetraspanin silencing in MM.

Co-administration of simvastatin and cytotoxic drugs is advantageous in myeloma cell lines.

We have evaluated the potential application of simvastatin (Sim) combined with conventional cytotoxic drugs for the treatment of multiple myeloma. RPMI 8226 and U266 myeloma cells seeded in culture plates were treated with Sim (5 and 10 microM, respectively) combined with melphalan (Mel; 25 and 20 microM, respectively) or dexamethasone (Dex; 1 microM). We assessed cell cycle (propidium iodide staining and flow cytometric analysis), cell morphology, viability (WST1), total cell count and cell death (Trypan blue exclusion). Sim significantly enhanced the anti-myeloma activity of cytotoxic agents in vitro (p<0.05). Incubation of U266 and RPMI 8226 with Sim prior to Mel increased the cytotoxicity in an additive manner, whereas the exposure of U266 to combined Sim and Dex resulted in a synergistic amplification of the individual effects. Combined application of Dex and Sim to RPMI 8226 cells resulted in antagonistic activity. The possible roles of Ras and phosphoinositol 3-kinase are discussed.

Microenvironment factors do not afford myeloma cell lines protection from simvastatin.

BACKGROUND: The intensive interactions of myeloma cells (multiple myeloma, MM) with microenvironmental components of the bone marrow contribute significantly to their proliferation and survival. It has been shown that these signals confer drug resistance, delineating their circumvention as a primary objective in disease treatment. This study was designed to assess the effect of some major extracellular factors on the previously established anti-neoplastic response of myeloma cells to simvastatin (Sim). STUDY DESIGN: RPMI8226, U266, and ARH77 seeded in culture plates precoated with fibronectin (FN)/agarose/none were treated with Sim, insulin-like growth factor-I (IGF-I), interleukin-6 (IL-6) or combinations for 5 d. Then we assessed cell morphology, viability (WST1), cell cycle (propidium iodide, PI, staining and flow cytometric analysis), total cell count, and cell death (trypan blue exclusion), and DNA fragmentation. RESULTS AND CONCLUSIONS: Reduced viability was demonstrated with Sim in all treated cell lines with and without co-administration of IGF-I or IL-6 (P < 0.05). The extent of inhibition did not vary between Sim only and combinations (NS). FN did not influence cell response to Sim alone or combined with IL-6/IGF-I (NS). We conclude that IL-6, IGF-I, and FN do not afford myeloma cell lines protection from Sim modulation.

The effects of doxorubicin on apoptosis and adhesion molecules of normal peripheral blood leukocytes-an ex vivo study.

This ex vivo study was designed to evaluate the effect of doxorubicin (Dox) on normal peripheral blood leukocytes (PBL) in terms of apoptosis and membranal expression levels of adhesion molecules. Blood was drawn immediately prior to and after Dox administration from 21 breast cancer patients, and incubated at room temperature for 24 h. Flow cytometry was employed in analysis of apoptosis with Annexin-V and protein membranal expression levels with monoclonal antibodies to CD49d, CD18, CD11a-c and CD63. Dox induced statistically significant apoptosis in all three major PBL subpopulations (p<0.01). Between 70 and 90% of samples underwent apoptosis in all PBL subgroups. No significant change was observed in the membranal level of CD63, CD49d and CD11a-c after chemotherapy in any PBL subpopulation. However, a significant reduction in the membranal level of CD18 was demonstrated in polymorphonuclear cells after Dox (p<0.005) both in apoptotic and non-apoptotic cells (p<0.05), suggesting a direct effect of Dox rather than an apoptosis-associated phenomenon. We observed the expected leukopenia 10 days after Dox administration with no correlation to apoptosis, suggesting that leukopenia by Dox is largely attributed to toxicity of blood progenitors.

The antiapoptotic effects of blood constituents in patients with chronic lymphocytic leukemia.

OBJECTIVE: Clonal B-lymphocytes of chronic lymphocytic leukemia (B-CLL) are characterized by decreased sensitivity to programmed cell death and, therefore, they accumulate in vivo. However, these malignant cells die rapidly in vitro. In the current study we concentrated on the contribution of autologous serum (AS) and lymphocyte subsets to the survival of the malignant cells in vitro. METHODS: Mononuclear cells from the peripheral blood of 26 CLL patients and 24 controls were incubated overnight in the presence or absence of AS and heat-inactivated AS (HI-AS) or fetal calf serum (FCS). Also, isolated B cells were incubated at different concentrations in the presence of AS and/or isolated T cells. The level of apoptosis of CD19+ cells was measured by flow cytometry. RESULTS: Spontaneous apoptosis of unfractionated B-CLL cells incubated with AS, FCS or without serum was significantly lower than the rate of B-cell death in the control group, in similar culture conditions. AS had an antiapoptotic effect on unfractionated B-CLL cells when compared with FCS. The rate of apoptosis of B-CLL cells was directly associated with stage. HI of AS had a variable effect, which was related to the stage of the disease. High concentrations of B cells and the addition of autologous T cells reduced the rate of apoptosis when incubated without serum. The antiapoptotic effect of T cells was most prominent in progressive stages. CONCLUSIONS: B-CLL cells exhibit decreased spontaneous apoptosis, which is partially prevented by humoral (AS) and cellular (T cells and B-CLL cells) factors. The equilibrium between apoptotic and antiapoptotic factors changes with disease progression.

Thalidomide down-regulates transcript levels of GC-rich promoter genes in multiple myeloma.

Thalidomide (Thd), a potent teratogen, was shown to have therapeutic potential in cancer, primarily in multiple myeloma (MM), yet its mechanism of action has not been elucidated. It was recently suggested that its teratogenicity is derived from interference in expression of genes regulated by GC-rich promoters by blocking the binding of SP1 transcription factor to its motif. We explored the validation of the proposed model by focusing on potential molecular targets associated with MM pathogenesis. Cell lines RPMI 8226, U266, and ARH-77 were exposed for 24 h to racemic Thd and analyzed for apoptosis, membranal expression of CD29 and CD63, transcript level of hTERT, CD63, and IGFI-R (characterized by GC-rich motifs) and telomerase activity. Analysis of an hTERT core promoter reporter gene expression [enhanced green fluorescent protein (EGFP)] in transiently transfected RPMI 8226 incubated with racemic and steric (+/-)-enantiomers of Thd was performed. A consistent reduction ( approximately 10-40%) in transcript levels of all three assayed genes in all three cell lines was demonstrated in the presence of racemic Thd. Significant reduction of EGFP was demonstrated in cells transfected with hTERT reporter gene and treated with racemic and (S)-Thd. Our results show that Thd's antimyeloma activity can be ascribed to the same mechanism responsible for its teratogenic effect and that the inhibition of GC-rich promoter genes is mostly attributed to the S-racemate. Indeed, this selectivity delineates GC-rich promoter genes as a unique group eligible for specific drug targeting.

Granulocyte colony-stimulating factor administration upregulates telomerase activity in CD34+ haematopoietic cells and may prevent telomere attrition after chemotherapy.

Hematopoietic reconstitution could be associated with premature ageing of the transplanted cells and a high frequency of myelodysplastic syndrome and secondary leukaemia. Telomere length decreases with cell divisions and age, and at a crucial length it is associated with chromosomal instability and cell senescence. Telomerase is a reverse transcriptase enzyme that adds nucleotides to chromosomal ends. Most somatic cells lack telomerase activity yet haematopoietic stem cells retain low levels of telomerase. Some studies have found that chemotherapy and stem cell transplantation lead to the accelerated shortening of telomere length. As granulocyte colony-stimulating factor (G-CSF) is routinely used in the mobilization of stem cells for transplantation, we evaluated its effects on telomerase activity and regulation, and on telomere dynamics, in normal donors and selected lymphoma patients. Administration of G-CSF increased telomerase activity in CD34+ haematopoietic cells compared with controls. In marrow-derived CD34+ cells, telomerase activity increased sevenfold, compared with a 14-fold increase in peripheral-blood-mobilized CD34+ cells. A parallel increase in the expression of human telomerase enzyme reverse transcriptase RNA and protein kinase C alpha occurred. In addition, G-CSF administration to five lymphoma patients after consecutive courses of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy, resulted in telomere length preservation or elongation, as opposed to marked attrition in patients who did not receive growth factors. We conclude that the in vivo administration of G-CSF prevents or attenuates telomere attrition associated with chemotherapy administration. This attenuation may contribute to the preservation of telomere integrity inG-CSF-primed transplanted stem cells.

Initial exposed phosphatidylserine levels correlate with cellular response to cytotoxic drugs.

Phosphatidylserine's (PS) membranal distribution is associated with an expanding variety of biological processes. We studied the relevance of preliminarily exposed membranal PS levels to cellular effects of cytotoxic agents. PBL of normal controls (n = 18) and patients with doxorubicin-treated breast carcinoma (n = 27) or 5'-fluorouracil-treated colorectal cancer (n = 32) were assayed before and after drug infusion. Membranal expression levels of PS, adhesion molecules (CD18, CD11a-c, CD63) and Fas-R of leukocyte subtypes were assessed by flow cytometer. Statistical analysis was implemented. Our results demonstrate external expression of PS on all leukocyte subpopulations despite non-apoptotic light scatter characteristics. Several distinct features were observed of which the more prominent were: leukocyte subtypes each display characteristic PS levels; cancer patients' PBL display higher preliminary PS levels than normal controls in all cell groups; and existence of negative correlations between initial membranal PS levels and drug-induced changes in its expression. Our findings underscore the complex involvement of PS in PBL apoptosis and possibly drug resistance.