Sibling cord blood donor program for hematopoietic cell transplantation: the 20-year experience in the Rome Cord Blood Bank.

Umbilical cord blood (UCB) represents a source of hematopoietic stem cells for patients lacking a suitably matched and readily available related or unrelated stem cell donor. As UCB transplantation from compatible sibling provides good results in children therefore directed sibling UCB collection and banking is indicated in family who already have a child with a disease potentially treatable with an allogeneic hematopoietic stem cell transplantation. Particularly, related UCB collection is recommended when the patients urgently need a transplantation. To provide access to all patients in need, we developed a "Sibling cord blood donor program for hematopoietic cell transplantation". Here we report results of this project started 20years ago. To date, in this study a total of 194 families were enrolled, a total of 204 UCB samples were successfully collected and 15 pediatric patients have been transplanted. Recently, some authors have suggested novel role for UCB other than in the transplantation setting. Therefore, future studies in the immunotherapy and regenerative medicine areas could expand indication for sibling directed UCB collection.

Evaluation of the prognostic relevance of L-selectin and ICAM1 expression in myelodysplastic syndromes.

OBJECTIVES: An aberrant pattern of expression of L-selectin and intercellular adhesion molecule 1 (ICAM1) may characterise CD34+ blast cells in myelodysplastic syndromes (MDS) and secondary acute myeloid leukaemia (sAML). METHODS: In a three-colour flow cytometric assay, we evaluated the expression of L-selectin and ICAM1 on CD34+ blast cells from the bone marrow (BM) of 66 MDS patients; for the purpose of comparison CD34+ blast cells of 18 sAML and CD34+ stem cells of 17 normal donors were also analysed. RESULTS: The ratio of L-selectin/ICAM1 expression was identified as a parameter correlated with the percentage of BM blast infiltration and the time to leukaemic progression among MDS patients. In fact, the values of L-selectin/ICAM1 ratio were inversely correlated with the BM blast infiltration (r = -0.34, P = 0.004). Furthermore, MDS patients with a baseline ratio <1 had a higher leukaemic progression rate (41% vs. 19%, P = 0.008); the actuarial risk of disease progression for this subgroup of MDS patients was also higher (64% vs. 11% at 2 yr, P = 0.002). Furthermore, in two patients a decrease of the ratio was observed when overt leukaemic transformation occurred; conversely, restoration of a normal ratio was observed in two patients after a chemotherapy-induced remission. CONCLUSION: (i) L-selectin is defective in the stem cell compartment of MDS and sAML, whereas ICAM1 is overexpressed; (ii) the ratio of their expression has a prognostic role; and (iii) a ratio <1 significantly predicts progression to overt leukaemia in MDS patients.

Monitoring of minimal residual disease in adult acute myeloid leukemia using peripheral blood as an alternative source to bone marrow.

BACKGROUND AND OBJECTIVES: To date, bone marrow (BM) is the most common source of cells to use in order to assess minimal residual disease (MRD) in acute myeloid leukemia (AML). In the present study, we investigated whether peripheral blood (PB) could be an alternative source of cells for monitoring MRD in AML. DESIGN AND METHODS: Fifty patients with AML were monitored for MRD after the achievement of complete remission. Using multiparametric flow cytometry we compared the levels of MRD in 50 and 48 pairs of BM and PB after induction and consolidation, respectively. RESULTS: After induction and consolidation therapy, the findings in BM and PB were significantly concordant (r=0.86 and 0.82, respectively, p<0.001 for both comparisons). The cut-off value of residual leukemic cells in PB which correlated with outcome was 1.5x10 (-4). Thirty-three of 43 (77%) patients with >1.5x10 (-4)residual leukemic cells in PB after induction had a relapse, whereas the seven patients with lower levels did not (p=0.0002). After consolidation, 38 patients had a level of MRD >1.5x10 (-4)and 31 (82%) had a relapse; nine out of the remaining ten patients, whose levels of MRD were below 1.5x10 (-4), are still relapse-free (p=0.00006). In multivariate analysis, PB MRD status at the end of consolidation was found to have a significant effect on relapse-free survival (p=0.036). INTERPRETATION AND CONCLUSIONS: These preliminary results indicate that: (i) PB evaluation can integrate BM assessment for MRD detection in patients with AML; (ii) PB MRD status at the end of consolidation therapy may provide useful prognostic information.

Combined analysis of bcl-2 and MDR1 proteins in 256 cases of acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: The objective of this study was to investigate the expression of MDR1 and bcl-2 proteins in de novo acute myeloid leukemia (AML). DESIGN AND METHODS: The expression of MDR1 and bcl-2 was analyzed by flow cytometry in a large series of 256 consecutive cases of AML. The results were recorded as percentage of positivity and relative mean fluorescence intensity (rMFI). To determine individual protein levels, an index which equals the product of the percentage of positive cells and rMFI was generated. RESULTS: Using cut-offs of >or=800 and 300 of the index value for bcl-2 and MDR1 expression, respectively, we identified 4 different classes of AML: 1) double negative; 2) single positive bcl-2+/MDR1-; 3) single positive bcl-2-/MDR1+; 4) double positive. The highest incidence of double negative cases was observed in the M2 class whereas double positive cases occurred more frequently in the M4, M5 and M6 subgroups. Seventy-eight percent and 71% of M0 and M1, respectively, showed single positive bcl-2+/MDR1- expression (p = 0.00001). Twenty-eight percent of patients belonging to the double positive category achieved complete remission, whereas for double negative, single positive bcl-2+MDR1- and single positive bcl-2-/MDR1+ category, the complete remission rate was 69%, 52% and 56%, respectively (p = 0.00038). In multivariate analysis, the double positive status independently affected frequency of complete remission (p = 0.008). INTERPRETATION AND CONCLUSIONS: Bcl-2 is over-expressed in CD34+ AML; conversely, MDR1 is over-expressed in CD34- AML. However, the combined expression of the two proteins defines a subset of AML with a very poor prognosis.

CD90/Thy-1 is preferentially expressed on blast cells of high risk acute myeloid leukaemias.

Different transformation mechanisms have been proposed for elderly acute myeloid leukaemia (AML) and secondary AML (sAML) when compared with de novo AML or AML of younger patients. However, little is known regarding differences in the immunophenotypic profile of blast cells in these diseases. We systematically analysed, by flow cytometry, 148 patients affected by de novo (100 cases) or sAML (48 cases). By defining a cut-off level of 20% of CD34+ cells co-expressing CD90, the frequency of CD90+ cases was higher in sAML (40%) versus de novo AML (6%, P < 0.001), elderly AML (>60 years) (24%) versus AML of younger patients (10%, P = 0.010) and poor- versus good-risk karyotypes (according to the Medical Research Council classification, P < 0.001). The correlation between CD90 expression, sAML and unfavourable karyotypes was confirmed by analysing the subset of CD34+ AML cases alone (91/148). Consistently, univariate analysis showed that expression of CD90 was statistically relevant in predicting a shorter survival in CD90+ AML patients (P = 0.042). Our results, demonstrating CD90 expression in AML with unfavourable clinical and biological features, suggest an origin of these diseases from a CD90-expressing haemopoietic progenitor and indicate the use of CD90 as an additional marker of prognostic value in AML.

Comparison between conventional banding analysis and FISH screening with an AML-specific set of probes in 260 patients.

Fluorescence in situ hybridization (FISH) is becoming popular in the diagnosis of clonal chromosomal abnormalities. We set up a fast FISH procedure using an extensive set of specific probes. Conventional banding analysis (CBA) and FISH were compared in 260 newly diagnosed acute myeloid leukemia (AML) patients. For FISH the following probes were used: MLL, CBF-beta/MYH11, ETV-6/AML1; AML1/ETO, BCR/ABL, PML/RAR, c-MYC, TP53, RB1, 5q31/5p15.2, 5q33-34, 7q31/CEP7, 20q13; CEP 4, X, Y. Result time was 96 h for CBA versus 5 h for FISH from direct harvest. CBA showed clonal abnormalities in 41% (n=105/260), normal karyotype in 39% (n=102/260) and failed in 20% (n=53/260). FISH screened all patients and detected abnormalities in 39% (n=102/260); CBA and FISH together identified abnormalities in 49% (n=128/260). In six patients with normal CBA and in eight patients with clonal karyotype, it detected further cryptic abnormalities. CBA showed clonal abnormalities in 13% of patients negative at FISH (n=21/158). FISH screening does not add relevant information to CBA, but is the quickest method for detecting major genetic abnormalities in AML. The speed of FISH is very valuable in AML-M3/M3v because PML/RAR+ patients require specific therapy. Furthermore, we suggest FISH screening in failed, complex or suboptimal quality chromosome and specific FISH analysis for 5q, 7q, 12p, 17p, inv(16), t(11q23) in order to implement CBA accuracy.

P-glycoprotein and BCL-2 levels predict outcome in adult acute lymphoblastic leukaemia.

Concurrent resistance mechanisms, such as P-glycoprotein (PGP) and bcl-2, may contribute to a worse outcome in adult acute lymphoblastic leukaemia (ALL). Between 1990 and 2000, we analysed PGP and bcl-2 by flow cytometry, using two anti-PGP (C219 and JSB-1) monoclonal antibodies (mAbs) and an anti-bcl-2 mAb in 115 de novo adult ALL patients. Both a longer overall survival (OS) and longer disease-free survival (DFS) were observed in PGP-negative patients (23%vs 0% at 3 years, P = 0.011 and 29%vs 0% at 2 years, P = 0.006 for C219 respectively; 42%vs 0% at 1.5 years, P = 0.004 and 53%vs 0% at 8.5 months, P = 0.00006 for JSB-1 respectively). Bcl-2 positivity was associated with a significantly higher complete remission rate (90%vs 66%, P = 0.01). Moreover, in 69 patients not presenting with either t(9;22) or B-mature immunophenotype, PGP negativity (JSB-1) maintained its significant favourable prognostic impact with regard to OS (41%vs 0% at 1.5 years, P = 0.009) and DFS (83%vs 0% at 6 months, P = 0.0005). Importantly, within a subset of 62 patients with normal (n = 31) or unknown (n = 31) karyotype, PGP (JSB-1)-negative patients showed both a significantly longer OS and DFS (63%vs 0% at 1.4 years, P = 0.018 and 84%vs 0% at 6 months, P = 0.001 respectively). In multivariate analysis, JSB-1 (P = 0.008) and cytogenetics (P = 0.02) were found to be independent prognostic factors with regard to DFS. Therefore, in adult ALL, PGP and bcl-2 represent sensitive indicators of clinical outcome, and potential targets of novel molecules aimed at overcoming chemoresistance and recurrent relapses.

Multidimensional flow cytometry for detection of minimal residual disease in acute myeloid leukemia.

The term minimal residual disease (MRD) describes the situation in which, after chemotherapy for acute leukemia (AL), a morphologically normal bone marrow (BM) can still harbor a relevant amount of residual malignant cells. Several techniques are now amenable to investigate MRD, and all together they have designated a new era in which a re-definition of the current criteria of complete remission (CR) is required. Depending upon the measured level of MRD we can distinguish a variety of clinical situations ranging from a potentially cured disease to short-term remission. In the context of this spectrum of conditions there would be room for different therapeutic strategies ranging from no further therapy to pre-emptive therapy to treat early relapses (immunologic and/or molecular relapses). This review will focus on the state of art of MRD detection in acute myeloid leukemia (AML) using multidimensional flow cytometry (MFC), and will cover the laboratory and clinical aspects of this approach.

Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML).

The inability to undergo apoptosis is a crucial mechanism of multidrug resistance in acute myeloid leukemia (AML), and the analysis of mitochondrial apoptotic proteins may represent a significant prognostic tool to predict outcome. Bcl-2 and Bax oncoproteins were evaluated in 255 de novo AML patients (pts) by flow cytometry using an anti-bcl-2 monoclonal antibody (MoAb) and an anti-bax MoAb. The results were expressed as an index (bax/bcl-2) obtained by dividing bax mean fluorescence intensity (MFI) and bcl-2 MFI. Lower bax/bcl-2 ratio was associated with French-American-British (FAB) M0-M1 classes (P =.000 01) and CD34 more than 20% (P <.000 01). There were striking inverse correlations between CD34 or CD117 MFI and bax/bcl-2 values (r = -.40, P <.000 001 and r = -.29, P =.000 002), confirming that immaturity is consistent with this index. Moreover, lower bax/bcl-2 levels were correlated with poor-risk cytogenetics (P =.0002). A significant higher complete remission (CR) rate was found in pts with higher bax/bcl-2 levels (79% versus 45%; P =.000 01). Also, both a longer overall survival (OS) and disease-free survival (DFS) were observed in pts with higher bax/bcl-2 levels (P =.000 01 and =.019). Noteworthy, bax/bcl-2 levels accurately predicted the clinical response and outcome of pts with normal or unknown cytogenetics. Indeed, within this subset of 147 pts, higher bax/bcl-2 ratio was significantly associated both with a higher CR rate (86% versus 42%; P <.000 01) and a longer OS (P =.0016). The independent prognostic value of bax/bcl-2 ratio was confirmed in multivariate analysis. Therefore, mitochondrial oncoproteins, such as bcl-2 and bax, represent both sensitive indicators of clinical outcome and potential targets of novel proapoptotic molecules in order to circumvent chemoresistance.

Autologous stem-cell transplantation for patients with acute myeloid leukemia aged over 60 yr.

OBJECTIVES: Preliminary reports have suggested that autologous stem-cell transplantation (ASCT) is feasible in elderly patients with acute myeloid leukemia (AML). The objective of this study was to describe the disease characteristics and treatment results from a series of 22 elderly AML patients undergoing ASCT. METHODS: The median age was 64 yr (range 61-71). Twenty patients were in first complete remission (CR1), two in CR2, and all were in performance status 0-1. The median interval between CR achievement and ACST was 3 months (range 2-5). In 20 cases peripheral blood stem cells were infused, in two bone marrow. RESULTS: All patients had a successful engrafment. One patient (5%) died from transplant-related complications. The median number of days to granulocytes > 500 mm-3 and platelets > 20 000 mm-3 was 11(range 9-15) and 13 (range 9-20), respectively. Non-hematologic toxicity included WHO grade III-IV stomatitis in 32% patients and grade IV nausea and vomiting in one (4.5%). Seven patients had fever of unknown origin, while in 14 a documented infection was diagnosed. Median duration of hospitalization was 31 d (range 16-60). CONCLUSIONS: After a median follow-up of 12 months from ASCT, nine patients are alive in continuous CR and 13 died from AML relapse. Median survival from diagnosis and disease-free survival (DFS) was 19 and 14 months, respectively. Our data show that ASCT with a standard conditioning regimen is feasible in AML patients aged more than 60 yr. Toxicity and hemopoietic recovery do not substantially differ from those observed in young adults. DFS and overall survival (OS) duration are encouraging, but a longer follow up is needed on a larger series of patients.

Positive selection of CD34+ cells by immunoadsorption: factors affecting the final yield and hematopoietic recovery in patients with hematological malignancies and solid tumors.

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 paticnts affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderlinc influence on the recovery of CD34+ cells. Forty patients received a median of 5 x 10(6) CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l in a median of 10 days whereas a PLT count above 20 x 10(9)/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide. was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content.

Clinical relevance of minimal residual disease detection in adult acute myeloid leukemia.

We have used flow cytometry to quantify minimal residual disease (MRD) in 63 patients with acute myeloid leukemia (AML). No significant correlation was found between the level of MRD after induction and disease outcome. After consolidation, a threshold of 3.5 x 10(-4) residual leukemic cells divided the 57 evaluable patients into two distinct groups: the MRDCons(+) and the MRDCons(-) group, with a relapse rate of 81% (22/27) and 27% (8/30), respectively (p = 0.000035). Although not correlated with prognosis, the level of MRD after induction course affected the degree of cytoreduction achieved with consolidation. In fact, the patients who entered a MRDCons(-) status had a median number of leukemic residual cells of 1.8 x 10(-4) after induction; at the same stage, the bone marrow of patients who were in a MRDCons(+) condition harbored a median level of 1.7 x 10(-3) malignant residual cells (p = 0.00073). The MRDCons(+) status also correlated significantly with poor/intermediate risk cytogenetics, MDR1 phenotype, short duration of overall survival, and relapse-free survival (p = 0.024, 0.021, 0.00001, and 0.00001, respectively). In multivariate analysis, the MRDCons(+) status was associated with a high probability of relapse (p < 0.00026) and short duration of relapse free survival (p = 0.008). Stem cell transplantation did not seem to alter the prognostic impact of high levels of MRD after consolidation: within the MRDCons(+) group, the relapse rate after transplant was 78%. Thus, a MRD > or = 3.5 x 10(-4) leukemic cells at the end of consolidation strongly predicts relapse, and is significantly associated with MDR1-positive phenotype and intermediate/unfavorable cytogenetics.