Adhesion receptors on peripheral blood leukemic B cells. A comparative study on B cell chronic lymphocytic leukemia and related lymphoma/leukemias.

The expression of a series of adhesion receptors: L-selectins (CD62L): Leu-8, several integrins (LFA-1: CD11a/CD18, VLA-4: CD49d/CD29 and VLA-5: CD49e/CD29), ICAM-1(CD54) and the 'homing receptor' (CD44) were investigated by a dual color flow cytometry in 56 cases of B cell disorders namely, 39 chronic lymphocytic leukemias (CLL), four hairy cell leukemia (HCL), seven splenic lymphoma with villous lymphocytes (SLVL) and six other non-Hodgkin's lymphoma (NHL). The functional activity of L-selectins was assessed with L-selectin ligand analogs (polyphosphomonester core polysaccharide: PPME and fucoidin). Leukemic B cells were identified with phycoerythrin-conjugated monoclonal antibodies (McAbs) anti-CD19, anti-kappa/lambda investigated simultaneously for the expression of adhesion receptors estimated with fluorescein-isothiocyanate (FITC) conjugated McAbs. The percentage of leukemic cells expressing L-selectins (Leu-8) was high in CLL (52% of positive cases) and integrin expression (LFA-1, VLA-4, 5) was low (19 and 33%, respectively), while a reverse pattern, low Leu-8 (17%), and a high VLA-4 (77%), was observed in non-CLL cases. The expression of LFA-1 alpha-chain was variable in non-CLL cases, and the LFA-1 heterodimer was expressed on most clonal B cell in NHLs (92%). LFA-1 alpha-chain was detected on cells from only one HCL case, while beta2 integrin was regularly expressed on hairy cells. VLA-5 integrin was found on a relatively small number (26%) of mature B cell leukemias. A remarkable finding was the detection of ICAM-1 in all CLL cases albeit the number of positive cells was significantly lower (P < 0.05) compared to non-CLL cases. CD44 was expressed on a high number of neoplastic cells in all the investigated categories. There was no correlation between the expression of the adhesion molecules and clinical and laboratory parameters except for CD18 which was expressed on a significantly (P < 0.05) higher number of leukemic cells in CLL with more advanced stages. This study demonstrates that even closely related B cell leukemia/lymphomas have a certain well defined and strictly variable adhesion profile which is characteristic of the disease entity and therefore, the adhesion profile may offer additional information useful for differential diagnosis and study of disease pathogenesis.

The existence of lymphoid lineage restricted Philadelphia chromosome-positive acute lymphoblastic leukemia with heterogeneous bcr-abl rearrangement.

Analysis of lineage involvement was performed in 17 Philadelphia chromosome-positive acute lymphoblastic leukemia patients with no history of chronic myeloproliferative disorder. The percentage of blastic cells as defined by flow cytometry matched that of the Ph-positive cells in 14 out of 17 patients. The bcr-abl rearrangement was investigated by fluorescent in situ hybridization in morphologically identified blastic cells, myeloid elements, lymphocytes and erythroblasts using a combined light and fluorescent microscopical imaging. Lymphoid lineage restriction could be determined in all but three of the patients. These 14 patients exhibited heterogeneity in terms of m-bcr and M-bcr types of translocation as revealed by reverse transcription polymerase chain reaction. The three patients with multilineage involvement and M-bcr type of translocation reverted to chronic phase and the percentage of Ph-positive cells remained high. Thus, we could identify an uncommitted stem cell origin among Ph-positive ALLs only in those patients whose disease subsequently proved to be a lymphoid blastic crisis with clinically silent chronic phase.

Phenotypic and genotypic analyses of blastic cell population suggest that pure B-lymphoblastic leukemia may arise from myelodysplastic syndrome.

The case history of a 70-year-old man with myelodysplastic syndrome terminated into acute leukemia in 22 months is presented. The leukemic cells exhibited multifocal acid phosphatase positivity and expressed TdT, CD45, CD34 and HLA-DR but not myeloid, monocytic or megakaryocytic differentiation antigenes. The genotypic analysis revealed clonal immunoglobulin heavy chain gene rearrangement. These phenotypic and genotypic analyses of the blastic cell population suggest that myelodysplastic syndrome may transform to pure acute lymphoblastic leukemia of B-cell origin.

Silent Philadelphia chromosome: a distinct developmental stage in a philadelphia chromosome-positive chronic myeloproliferation?

In this paper, a patient is described who presented with peripheral blood and bone marrow features uncharacteristic of chronic granulocytic leukemia, which proved to be Philadelphia (Ph) chromosome-positive by metaphase and interphase cytogenetic analyses but lacked the p210 type of BCR/ABL fusion gene mRNA product by two different sensitive RT-PCR assays. In the course of the 32-month follow-up with a termination into a myeloblastic crisis, molecular investigations were performed four times. They indicated a constantly high rate of Ph positive cells and lack of BCR/ABL mRNA expression, except in the second investigation, when the patient showed reverse transcription polymerase chain reaction positivity with b3/a2 type of chimera, fusion gene mRNA expression, and a striking change in the bone marrow histology. Our findings might indicate that the dormant Ph chromosome state may exist not only at the primitive progenitor, but also at the entire peripheral blood cell compartment level.

Biotinylated L-selectin ligand analogs as cytochemical probes in cytospin preparations.

Amino-derivatives of L-selectin ligand analogs: phosphonoester core polysaccharide (PPME) and fucoidin were biotinylated with the use of biotinyl-N-succinimide ester, and these biotinylated analogs b-PPME and b-fucoidin were demonstrated as useful tools to investigate the functional activity of L-selectins in cytospin preparations obtained from healthy human donors and from patients with chronic lymphocytic leukemia (CLL). The avidin/biotin system adds a new alternative to the application of the L-selectin ligand analogs (PPME, fucoidin) which have been formerly used as fluoresceinated, solid phase or immobilized probes.

L-selectins revealed by immobilized analogue molecules on human peripheral blood lymphocytes.

The lymphocyte-endothelial interaction is initiated by selection type adhesion molecules on the surface of the lymphocytes (L-selectins) and by their carbohydrate ligands (addressins) in the glycocalyx of the endothelial cells. Under experimental conditions they can be substituted by analogue sugars, such as polyphosphonomonoester core polysaccharide and fucoidin. In this study, the expression of phosphomannosyl and fucoidin receptors is demonstrated on lymphocytes from human peripheral blood by polyacrylamide immobilized analogues. Based on adhesion and sugar inhibitory experiments, authors suggest that lineage and probably species specific differences exist in the expression and activity of the selectins responsible for binding of the two analogue molecules.

Expression of an adhesion molecule and homing in B-cell chronic lymphocytic leukaemia: II. L-selectin expression mediated cell adhesion revealed by immobilized analogue carbohydrates in B-cell chronic lymphocytic leukaemia and monoclonal lymphocytosis of undetermined significance.

The L-selectin mediated adhesion of freshly isolated peripheral blood mononuclear cells (PBMCs) to phosphonomonoester core polysaccharide (PPME) and fucoidin derivatized gels was investigated in seven cases of monoclonal lymphocytosis of undetermined significance (B-MLUS) and 12 cases of chronic lymphocytic leukaemia: B-CLL, patients with peripheral lymphocytosis (LY-patients), lymph node enlargement (LN-patients) and splenomegaly (SM-patients). PBMCs isolated from the peripheral blood of 10 healthy donors served as controls. The binding to PPME and fucoidin correlated well (n = 19, P = 0.01). Adhesion of PBMCs from B-MLUS and B-CLL showed a greater variability than controls. A higher number of cells, on average, bound to PPME and fucoidin derivatized polyacrylamide gels in B-MLUS than in B-CLL. However, the differences observed were not statistically significant. In four cases with B-CLL, the stimulatory effect of interferon-alpha on the function of L-selectin and some other accessory molecules was also studied. The increased binding of PBMCs to immobilized analogue molecules (PPME, fucoidin) and to high endothelial venules (HEVs) in the in vitro HEV-binding assay supports the notion that interferon-alpha not only increases the expression of the adhesion molecules, but also results in an enhanced adhesive function.

Changes in adhesion molecule expression and function in B-cell chronic lymphocytic leukaemia after in vitro interferon-alpha stimulation.

Peripheral blood mononuclear cells (PBMCs) from 10 B-CLL patients were investigated after 24 hours of in vitro interferon-alpha (IFN-alpha) stimulation. The constitutional expression of the L-selectins (LECAM-1), LFA-1/CD11a, VLA alpha-4/CDw49d and ICAM-1/CD54 adhesion molecules was detected, and changes in their density after IFN-alpha stimulation were compared to results obtained by the high endothelial venule (HEV)-binding assay and a carbohydrate (phosphonomannan core polysaccharide: PPME and fucoidin) immobilization test. The LECAM-1 and ICAM-1 molecules were expressed on the great majority of CLL cells, while the LFA-1 and VLA-4 alpha-chains were expressed by only a small number of cells. Statistically significant changes (p < 0.001) were observed in LECAM-1 antigen density (changes in mean cell fluorescence), as well as in functional tests (HEV-, PPME- and fucoidin-binding; p < 0.01) after in vitro IFN-alpha stimulation. Based on a prior study (Jewell et al., Leukemia 1992: 6: 400-404) and on the present findings, not only an increased expression but also an enhanced function of the L-selectins seem to be well substantiated after IFN-alpha stimulation, which may explain the therapeutic effect of IFN-alpha in reducing the accumulation of leukaemic B cells in the blood. The remarkably high expression of ICAM-1 in this series necessitates further studies to clarify the exact expression rate and role of this molecule.

Expression and function of L-selectin molecules (LECAM-1) in B-cell chronic lymphocytic leukemia.

BACKGROUND: The notion that adhesion molecules play a crucial role in lymphoma/leukemia dissemination is widely accepted. Individual cases of B-cell chronic lymphocytic leukemia (B-CLL) show well-defined variables in the extent and pattern of peripheral blood and nodal involvement. The L-selectin adhesion molecule (TQ1/Leu-8, LAM series and LECAM-1) initiates the attachment of lymphocytes to the high endothelial venules (HEVs), and as a consequence the entrance of lymphocytes from the blood into the peripheral lymph node (recirculation which may be operative in lymphoma/leukemia dissemination as well). MATERIALS AND METHODS: The constitutional expression of L-selectin molecules (LECAM-1) on peripheral blood mononuclear cells (PBMCs) from B-CLL (16 cases) was examined and correlated with receptor function in an HEV-binding assay and in a ligand immobilization test. RESULTS AND CONCLUSIONS: A correlation was found between constitutional expression and function of the L-selectins, namely the higher the number of cells expressing L-selectin molecules at a measurable level on the cell surface, the greater the number of cells showing attachment in the tests. It is suggested that many aspects of the biological and clinical heterogeneity of B-CLL will be explained by revealing the exact adhesion profile and function in different subtypes of the disease.

Laparoscopic radical prostatectomy: the initial UK series.

OBJECTIVE: To test the reproducibility of other series of laparoscopic radical prostatectomy (LRP) for safety, efficacy and early oncological and functional results. PATIENTS AND METHODS: One hundred consenting patients with clinically localized adenocarcinoma of the prostate and a Gleason sum of < or = 8 opting for surgery underwent LRP undertaken by one surgeon. Their mean (range) age was 62.2 (52-72) years, weight 78.8 (65-100) kg, prostate specific antigen (PSA) level 8.0 (2-32) ng/mL, and Gleason sum 6.0 (4-8). A five-port antegrade transperitoneal technique was used in all cases. RESULTS: The mean (range) operative duration was 245 (145-600) min, blood loss 313 (50-1300) mL, parenteral morphine sulphate administration 20.2 (0-160) mg and hospital stay after LRP 4.2 (3-13) nights. Bilateral neurovascular bundle preservation was attempted in 58% of patients. The transfusion rate was 3%. The conversion and re-intervention rates were 1% and 2%, respectively. There were eight complications, six of which were in the initial 26 cases, i.e. bladder neck stenosis (two), and rectal injury, laparotomy for bleeding, premature drain removal leading to urinary peritonitis, ulnar nerve neuropraxia, port-site hernia and paralytic ileus in one each. The positive surgical margin rate was 16%. All patients had a PSA level of < or = 0.1 ng/mL at 3 months. By 1 year 90% of patients were pad-free and 62% operated on using a bilateral nerve-sparing technique had erections. There were no biochemical failures. The mean (range) follow-up was 9.8 (1-24) months. CONCLUSION: The present results are similar to those reported by other centres with greater experience and confirm that LRP is an effective, safe and precise technique. Once intial experience has been gained it offers advantages over open surgery in the form of a dry and magnified operative site, and a lower likelihood of blood transfusion, in addition to the generic advantages of laparoscopy.

Transperitoneal or extraperitoneal laparoscopic radical prostatectomy: does the approach matter?

PURPOSE: : The greater accuracy of apical dissection and reconstruction in our first 100 patients undergoing transperitoneal laparoscopic radical prostatectomy (TLRP) was not matched by a proportionate increase in the rate of return to normal continence compared with our prior open prostatectomy experience. We postulated that greater bladder dysfunction due to the almost total bladder dissection mandated by TLRP might be responsible and this might be rectified by the adoption of laparoscopic radical prostatectomy using an extraperitoneal approach (ELRP). MATERIALS AND METHODS: : A total of 100 patients undergoing TLRP were compared with 100 undergoing ELRP. The groups were subdivided into halves to investigate the influence of any learning curve effect. All patients had clinical stage T3aN0M0 or less prostate cancer and they were operated on by a single surgeon. RESULTS: : Mean operative time (238.9 vs 190.6 minutes), blood loss (310.5 vs 201.5 ml), postoperative hospitalization (3.8 vs 2.6 nights) and catheterization duration (11.3 vs 10.1 days) were significantly greater in the TLRP group. After the first 50 cases were excluded in each group statistical significance persisted only for operative time (218.3 vs 184.2 minutes) and hospitalization (3.5 vs 2.5 nights). The pad-free rate was significantly lower 3 months following ELRP (80% vs 56%, p = 0.02). The overall 12-month pad-free rate for TLRP and ELRP was 90% and 96%, respectively. The overall 12-month erection rate for TLRP and ELRP was 61% and 82%, respectively. CONCLUSIONS: : ELRP is superior to TLRP with respect to operative time, hospitalization and early continence.

Philadelphia chromosome and/or bcr-abl mRNA-positive primary thrombocytosis: morphometric evidence for the transition from essential thrombocythaemia to chronic myeloid leukaemia type of myeloproliferation.

AIMS: The incidence, bone marrow morphology and genetic features of bcr+ essential thrombocythaemia were investigated. METHODS AND RESULTS: Sixty-four consecutive patients meeting the criteria of essential thrombocythaemia have been investigated for bcr-abl rearrangement and chimera mRNA expression. Reverse transcriptase-polymerase chain reaction indicated bcr-abl expression in six patients, in two of whom large fraction of the blood and bone marrow cells proved to be positive for Philadelphia chromosome (Ph) by fluorescent in-situ hybridization (FISH) and conventional cytogenetic analysis. In the remaining four patients FISH analysis could not detect Ph+ cells among the blood cells, but in one of these four patients conventional cytogenetic analysis indicated a very small fraction (2%) of Ph+ mitoses in the bone marrow (bcr+ essential thrombocythaemia patients). In three of these four patients, X-chromosome-linked clonality assay showed that the disease is of uncommitted stem cell origin. During an average of 57 month long follow-up no transformation to chronic myeloid leukaemia type of disease or acceleration/blastic crisis could be observed in the four bcr+ essential thrombocythaemia patients. They did not differ significantly from typical essential thrombocythaemia patients in quantitative indices of bone marrow cellularity or the size of megakaryocytes. In these two parameters as well as in the total nucleolus organizer region area per nucleus, however, significant differences could be detected between these four as well as typical chronic myeloid leukaemia patients. Statistical analysis of the morphometric data obtained from all six Ph+ and bcr+ essential thrombocythaemia patients combined indicated a shift of the bone marrow morphology towards the chronic myeloid leukaemia type of myeloproliferation. CONCLUSIONS: These investigations indicate that bcr+ essential thrombocythaemia is infrequent among essential thrombocythaemia patients, and this condition resembles essential thrombocythaemia more than chronic myeloid leukaemia. Various expansions of the Ph+ clone appear to lead to either essential thrombocythaemia or, rather, chronic myeloid leukaemia type of myeloproliferation; however, data in the present study do not indicate that bcr+ essential thrombocythaemia would be a form fruste variant of chronic myeloid leukaemia.