Exercise capacity and circulating endothelial progenitor cells in hemodialysis patients.

Mobilization of circulating endothelial progenitor cells (EPCs) is increased after acute exercise and training. This study aims to evaluate whether, in a low performance population, EPC levels may be related to exercise capacity in steady state conditions. Study population consisted of sixteen hemodialysis patients. The distance walked in the 6-minute walking test (6 MWD) and the maximal speed attained in an incremental treadmill test were used to assess the exercise capacity. Physical functioning was measured by the scale on the SF36 questionnaire. Quantification of peripheral blood CD34(+) cells and enumeration of EPCs, assessed as CD34(+) cells coexpressing AC 133 and vascular endothelial growth factor receptor-2, were performed. Hemoglobin concentration, white blood cells, high-sensitivity C-reactive protein, total cholesterol, and triglycerides were measured. Statistical analysis examined the relationship between blood progenitors cells versus performance parameters, laboratory parameters, age, body mass index, hemodialysis duration, and erythropoietin therapy. Univariate analysis revealed a significant association between percentage values of EPC and performance parameters only: 6 MWD (r=0.720; p=0.0017), maximal treadmill speed (r=0.721; p=0.0016), and physical functioning score (r=0.506; p=0.0453). A similar statistical association between EPC absolute values and performance parameters was found. No correlation between CD34 (+) and any parameter under study was observed. Multivariate analysis indicated 6 MWD as the most significant independent factor associated with EPC level. EPC percentage value was significantly lower (p=0.0087) in the worse (6 MWD < 300 m, n=8) than in the better performing group (6 MWD > 300 m, n=8). In a group of renal patients, mobilization of EPCs was related to the degree of exercise capacity, suggesting a possible connection with the cardiovascular risk in low performance populations limited by chronic diseases.

Functional and immunophenotypic characteristics of isolated CD105(+) and fibroblast(+) stromal cells from AML: implications for their plasticity along endothelial lineage.

BACKGROUND: In vitro cultures of BM cells from newly diagnosed patients with AML displayed a defective BM stromal compartment, with a reduced number of fibroblast-colony-forming unit (CFU-F: 1 +/- 1.25 SD) and a decreased proliferative ability. The purposes of our study were: 1). to select BM mesenchymal stem cells (MSC) and BM-derived stromal cells (BMDSCs) from AML patients at diagnosis and from healthy subjects, using an immunomagnetic system and either anti-CD105 or anti-fibroblast MAbs; 2). to study the immunophenotypic and functional properties of freshly isolated and cultured mesenchymal cells; 3). to test the in vitro plasticity of the selected cells to differentiate towards an endothelial phenotype. METHODS: Fresh mononuclear cells obtained from BM of 20 patients newly diagnosed with AML and from eight healthy subjects were selected by using anti-fibroblast and anti-CD105 MAbs. Freshly isolated cells were analyzed, characterized by flow cytometry using a wide panel of MAbs and seeded in long-term culture medium to assess CFU-F formation. The level of confluence after 30 days and functional capacity in a long-term colony-forming cell culture (LTC-CFC) were tested. Furthermore, the cultured selected cell populations were assayed for their ability to differentiate into an endothelial-like cell phenotype with the addition of vascular endothelial growth factor (VEFG) and endothelial cell growth supplement (ECGS). RESULTS: In normal subjects the selection produced an increase of the CFU-F number of 2.6-fold with anti-fibroblast MAb and 2.7-fold with the anti-CD105 MAb. Anti-fibroblast and anti-CD105 MAb selection from AML BM cells resulted in a statistically significant greater count of CFU-F that was respectively 10.6-fold (P = 0.04) and 14.4-fold (P = 0.00001) higher in comparison with the unselected AML samples. Interestingly, in 80% of AML samples immunoselection was also able to restore the capacity of the CFU-F to proliferate and form confluent stromal layers. The isolation of those layers sustained the proliferation and differentiation of hematopoietic stem cells in the LTC-CFC. The phenotypic profile of cultured BMDSCs was different from that of the freshly isolated cells, and changed in relation to the culture conditions: CD105+ selected cells cultured with VEGF and ECGS expressed endothelial markers, a finding that suggests that this cell subpopulation may have the potential to differentiate toward an endothelial-like phenotype. DISCUSSION: We report that immunomagnetic selection represents a valid tool for the selection of BM mesenchymal cells in samples obtained from both healthy subjects and patients with AML. This technique was able to rescue two functional and immunophenotypic compartments related to two different selected populations. In particular, the CD105+ cells isolated in AML displayed, after stimulation with VEGF and ECGS, the ability to change towards an endothelial-like cell phenotype, thus revealing an unexpected plasticity. Both CD105+ and fibroblast+ cells once successfully isolated might represent sources of mesenchymal cells populations useful for in vitro investigations and, above all, as therapeutic devices.

Partial deletions of long arm of chromosome 6: biologic and clinical implications in adult acute lymphoblastic leukemia.

Within 285 adult acute lymphoblastic leukemias (ALL) included in the multicenter GIMEMA 0496 trial and prospectively studied by conventional cytogenetics, 18 cases (6%) with long arm deletion of chromosome 6 (6q) were identified. These cases were divided into: (i) del(6q) only (n = 6); (ii) del(6q) plus other numerical and/or structural abnormalities (n = 8); (iii) del(6q) and other 'specific' translocations (n = 4). The biologic and clinical features of the patients carrying this anomaly, as well as their outcome, were compared with those of 267 patients without del(6q). A T cell phenotype was more frequently associated with del(6q) cases in general (P = 0.001) and particularly with cases presenting del(6q) as the isolated abnormality (P = 0.0027). No significant difference with respect to multidrug resistance (MDR)/P glycoprotein expression was observed between the two groups of patients (21% vs 28% of MDR-positive cases, respectively). A BCR-ABL fusion transcript was less frequently detected in cases with del(6q) (11%) compared with those without the anomaly (29%). p15 and p16 deletions were identified by Southern blot analysis in 21% of cases with del(6q) and in 26% of cases without del(6q). In this latter group, a T cell phenotype was less frequently associated with p15 and/or p16 deletion than in the group carrying del(6q) (36% vs 100% of cases, P = 0.011). Overall, patients with ALL and del(6q) had a high complete remission (CR) rate (83%); however, they had a lower 18 month event-free survival (31% vs 41%) and a higher relapse rate (70% vs 37%, P = 0.02) compared with patients without del(6q). To date, this is the largest series of adult ALL cases reported with del(6q) homogeneously treated, which have also been prospectively studied for MDR expression and for the detection of known fusion genes. This anomaly, as an isolated change, identifies a subset of cases with hyperleukocytosis (median WBC count 52 x 10(9)/l) and a strict correlation with a T cell phenotype. Overall, del(6q) seems to be associated with an unfavorable clinical outcome, although this finding will need to be confirmed by extended FISH analysis.

Preferential expression of the transcription coactivator HTIF1alpha gene in acute myeloid leukemia and MDS-related AML.

HTIF1alpha, a transcription coactivator which is able to mediate RARalpha activity and functionally interact with PML, is encoded by a gene on chromosome 7q32-34, which is a critical region in acute myeloid leukemias (AML). With the assumption that this gene may be related to AML, we investigated the HTIF1alpha DNA structure and RNA expression in leukemic cells from 36 M1-M5 AML patients (28 "de novo" and eight "secondary" to myelodysplastic syndrome (MDS)). Abnormal HTIF1alpha DNA fragments were never found, whereas loss of HTIF1alpha DNA was observed in the patients with chromosome 7q32 deletion and translocation, and in one case without detectable chromosome 7 abnormality. HTIF1alpha RNA was found in acute myelocytic leukemic blasts, and was almost undetectable in normal mononuclear cells. The expression varied among the patients: higher in M1 to M3 subtypes, with the highest values in M1; low levels were constantly observed in M4 and M5 AML. In addition, HTIF1alpha was significantly overexpressed in MDS-related AML (MDR-AML), but not in MDS. We also found that HTIF1alpha expression was high in myeloid cell lines. In myeloblastic HL60 and promyelocytic NB4 cells, induced to differentiate along the monocytic-macrophage pathway by TPA or vitamin D3, HTIF1alpha expression decreased, whereas it was maintained at high levels on induction to granulocytic differentiation by RA or DMSO. In K562 cells, HTIF1alpha RNA levels did not change after hemin-induced erythroid differentiation. These results suggest that HTIF1alpha could play a role in myeloid differentiation, being distinctly regulated in hematopoietic lineages.

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay.

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.

The prognostic value of cytogenetics is reinforced by the kind of induction/consolidation therapy in influencing the outcome of acute myeloid leukemia--analysis of 848 patients.

We studied the impact of cytogenetics and kind of induction/consolidation therapy on 848 adult acute myeloid leukemia (AML) patients (age 15-83). The patients received three types of induction/consolidation regimen: standard (daunorubicin and cytosine arabinoside (3/7); two cycles); intensive (idarubicin, cytosine arabinoside and etoposide (ICE), plus mitoxantrone and intermediate-dose Ara-C (NOVIA)); and low-dose (low-dose cytosine arabinoside). CR patients under 60 years of age, if an HLA-identical donor was available received allogeneic stem cell transplantation (allo-SCT); otherwise, as part of the program, they underwent autologous (auto)-SCT. CR rates significantly associated with 'favorable' (inv(16), t(8;21)), 'intermediate' ('no abnormality', abn(11q23), +8, del(7q)) and 'unfavorable' (del (5q), -7, abn(3)(q21q26), t(6;9), 'complex' (more than three unrelated cytogenetic abnormalities)) karyotypes (88% vs 65% vs 36%, respectively; P = 0.0001). These trends were confirmed in all age groups. On therapeutic grounds, intensive induction did not determine significant increases of CR rates in any of the considered groups, with respect to standard induction. Low-dose induction was associated with significantly lower CR rates. Considering disease-free survival (DFS), multivariate analysis of the factors examined (including karyotype grouping) showed that only age > 60 years significantly affected outcome. However, in cases where intensive induction was adopted, 'favorable' karyotype was significantly related to longer DFS (P = 0.04). This was mainly due to the favorable outcome of t(8;21) patients treated with intensive induction. Patients receiving allo-SCT had significantly longer DFS (P = 0.005); in particular, allo-SCT significantly improved DFS in the 'favorable' and 'intermediate' groups (P = 0.04 and P = 0.048, respectively). In conclusion our study could provide some guidelines for AML therapy: (1) patients in the 'favorable' karyotype group seem to have a longer DFS when treated with an intensive induction/consolidation regimen, adopted before auto-SCT instead of standard induction; this underlines the importance of reinforcement of chemotherapy, not necessarily based on repeated high-dose AraC cycles. Allo-SCT, independently of induction/consolidation therapy, should be considered an alternative treatment; (2) patients in the 'intermediate' karyotype group should receive allo-SCT; (3) patients in the 'unfavorable' karyotype group should be treated using investigational chemotherapy, considering that even allo-SCT cannot provide a significantly longer DFS, but only a trend to a better prognosis.

Detection of BCR/ABL rearrangements in adult acute lymphoblastic leukemia using a highly sensitive interphase fluorescence in situ hybridization method (D-FISH).

INTRODUCTION: One hundred-and-six adult cases of acute lymphoblastic leukemia were prospectively investigated using a highly sensitive interphase fluorescence in situ hybridization assay which utilizes DNA probes that detect a double BCR/ABL fusion signal (D-FISH) in cells carrying the t(9;22) to evaluate the reliability and specificity of this method for the detection of the Ph translocation. The results were compared with those obtained in the same cases by conventional cytogenetics and by reverse-transcription polymerase chain reaction. MATERIALS AND METHODS: The study was performed using DNA probes that span the common breakpoints of the t(9;22) translocation and that detect a double BCR/ABL fusion in cells carrying this karyotypic anomaly, one on the abnormal chromosome 9 and one on the Ph chromosome. RESULTS: Interphase D-FISH detected a high number of rearranged cases (22/106) compared to conventional cytogenetics (15/106) and RT-PCR (21/106). CONCLUSION: Interphase D-FISH emerges as a reliable, fast and relatively inexpensive tool for the detection of BCR/ABL rearrangements in adult ALL patients at diagnosis. It has a sensitivity clearly higher than conventional karyotyping and it may prove also superior to that of RT-PCR in cases with unusual BCR/ABL breakpoints. Our results suggest that D-FISH might be considered as the initial test for the diagnosis of Ph+ adult ALL.

BCL-1 rearrangements and p53 mutations in atypical chronic lymphocytic leukemia with t(11;14)(q13;q32).

BACKGROUND AND OBJECTIVES: The translocation t(11;14) (q13;q32), typically described in mantle cell lymphomas (MCL), has also been found in some cases of non-MCL lymphoproliferative disorders, such as splenic lymphoma with villous lymphocytes (SLVL), multiple myeloma (MM), prolymphocytic leukemia (PLL), typical and atypical chronic lymphocytic leukemia (CLL and aCLL). In order to define better the genetic features of aCLL with t(11;14), which could represent a distinct disease subset, we looked for genetic lesions in the BCL-1 locus and in BCL-2, BCL-6, c-myc and p53 genes. DESIGN AND METHODS: We investigated a panel of B-lymphoproliferative disorders with translocation t(11;14)(q13;q32) including nine aCLL, six MCL and one MM. Southern and Northern blot analysis was used to investigate DNA structure and RNA expression; SSCP and direct sequencing were used to detect and characterize p53 point mutations; cytofluorimetric analysis was used to quantify p53 protein. RESULTS: Alterations of BCL-2, BCL-6 and c-myc were not detected. Conversely, BCL-1 rearrangements were present in 4 out of 7 aCLL and in 2 out of 4 MCL. A high incidence of p53 gene alterations was found, almost equivalent in aCLL and MCL. INTERPRETATION AND CONCLUSIONS: Our results indicate that the occurrence of BCL-1 locus lesions in aCLL selected for t(11;14) is as high as in MCL. Interestingly, rearrangements in the mTC1 (minor translocation cluster 1) were only found in aCLL. Therefore, the two B-cell chronic lymphoproliferative disorders share similar molecular rearrangements and the t(11;14) identifies a subset of B-CLL sharing molecular features with MCL and characterized by aggressive clinical evolution.

Short term treatment with Escherichia coli recombinant human granulocyte-macrophage-colony stimulating factor prior to chemotherapy for Hodgkin disease.

BACKGROUND: Granulocyte-macrophage-colony stimulating factor (GM-CSF) administration stimulates the proliferation of hemopoietic progenitors. Shortly (48-96 hours) after its discontinuation, feedback phenomena occur and the progenitor proliferation rate drops below baseline levels. As the quiescence of hyperplastic bone marrow suggests that hemopoietic cells may be refractory to the toxic effects of cytostatic drugs, the decision was made to test the hypothesis that GM-CSF given before chemotherapy may be myeloprotective. METHODS: Fifty-six patients with newly diagnosed Stage II-IV Hodgkin disease, ages 18-77 years, were randomized to receive GM-CSF (5 microg/kg subcutaneously) or placebo from Day 7 to Day 4 before each chemotherapy administration (6 cycles of a hybrid of mechlorethamine, vincristine, procarbazine, and prednisone with doxorubicin, bleomycin, vinblastine, and dacarbazine). The treatment was considered a success if the delivery rate of chemotherapy was >90% after 3 cycles and >80% after 6 cycles. RESULTS: Thirty patients received GM-CSF and 26 placebo. The dose intensity (85.2% vs. 79.6%) and the overall success in terms of delivery rate (56.7% vs. 50%) were higher in the GM-CSF group, although these differences were not statistically significant. The neutrophil nadirs were higher in the GM-CSF group during the first three cycles and subsequently similar in both groups. CONCLUSIONS: No significant differences in terms of myelotoxicity or drug delivery were observed between the two treatment arms. Although the myeloprotective effect of the prechemotherapy administration of GM-CSF seems to be minimal, the data indicate a safe timing between GM-CSF discontinuation and further chemotherapy. Because cumulative myelotoxicity has been observed with other growth factors, given in the interval between the chemotherapy cycles, this may be relevant to the planning of rapid cycling.

A novel translocation t(1;7)(p36;q34) in three patients with acute myeloid leukaemia.

Studies of large numbers of patients have enabled the identification of relatively infrequent chromosome changes, such as inv(3)(q21;q26), t(6;9)(p23;q34) and t(8;16)(p11;p11), whose clinico-biological significance is gradually becoming clearer. Translocations involving chromosomes 1 and 7 are relatively rare in myeloid neoplasias, being found in far less than 1% of cases; the rearrangement that occurs most frequently consists of an unbalanced translocation [t(1;7)(p11; p11)], resulting in complete loss of 7q, associated with therapy-related or environmentally-induced high-risk myelodysplasia. We recently observed three cases of acute myeloid leukaemia (AML) with a previously unreported balanced translocation t(1;7) (p36;q34). Case 1 underwent autologous bone marrow transplantation and remains alive in CR; cases 2 and 3 relapsed after 10 and 4 months, respectively. The response to chemotherapy observed in our cases suggests that variable clinical features might be present in the broad cytogenetic category usually referred to as '7q abnormalities' and contributes to an interesting previous observation of prolonged disease-free survival in a subset of AMLs with 7q- as the isolated chromosome change.

Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells.

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34+ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples: in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 x 10(3) ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.

The effectiveness and tolerability of epoetin alfa in patients with multiple myeloma refractory to chemotherapy.

Anemia is a frequent complication of multiple myeloma, becoming chronic in patients who are resistant to chemotherapy. This randomized, parallel, controlled multicenter study (71 patients receiving concomitant chemotherapy) evaluated the efficacy and safety of epoetin alfa in improving anemia and eliminating the need for transfusions in multiple myeloma patients refractory to conventional first- or second-line chemotherapy. Forty patients were treated with subcutaneous epoetin alfa (150 IU/kg per dose, increasing to 300 IU/kg per dose, every 3 weeks) for 6 months, and 31 entered a control group. The epoetin alfa group had a significantly (P < or = 0.001) greater percentage of patients (75% vs. 21%) with increases in hemoglobin levels and/or reduced transfusion requirements. In 44 non pre-transfused patients (20 controls, 24 in the epoetin alfa group), the mean increase in hemoglobin was significantly (P < or = 0.0001) greater in the epoetin alfa group (+2.1 vs. -0.2 g/dl). Increases in hematocrit and red blood cells were also significantly (P < or = 0.0001) greater in epoetin alfa-treated patients, with corresponding reductions in transfusion requirement. In the 27 pre-transfused patients (11 controls, 16 in the epoetin alfa group), there was a trend towards reduced transfusional need in epoetin alfa-treated patients. Thus, in patients with multiple myeloma refractory to chemotherapy epoetin alfa is a well-tolerated treatment which improves anemia in non pre-transfused patients and appears to reduce transfusion need in those previously transfused.

Characterization of t(11;14) translocation in mantle cell lymphoma by fluorescent in situ hybridization.

Characterization of chromosome abnormalities in leukemia and lymphoma have contributed to the understanding of the molecular basis of these neoplastic diseases. In addition, specific chromosomal aberrations have acquired diagnostic or prognostic value. The t(11;14)(q13;q32) chromosome translocation has been detected in mantle cell lymphomas. However, possibly due to the limits of conventional cytogenetic analysis and the presence of different breakpoints at the molecular level, it is possible that the true percentage of association is underestimated. In our study, we used a yeast artificial chromosome, spanning the entire area where the rearrangements occur on chromosome 11q13, to detect the presence of translocations by fluorescent in situ hybridization experiments. We detected BCL-1 translocations in eight of eight patients with clinical and immunological features of mantle cell lymphoma, suggesting that the t(11;14) translocation is a critical event in the pathogenesis of MCL and may be a primary element for the diagnosis. Since this translocation is associated with poor prognosis, its detection may help to make a correct diagnosis as well as to evaluate residual disease, which is critical to plan a rational chemotherapy regimen.