A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117+ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients. Our results show that most of normal BM CD117+ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117+ cells are CD34+, these cells displaying a different FSC/SSC distribution when compared to the CD117+/CD34- cells. No CD117+/CD15+ and CD117+/CD10+ cells were detected and very few CD117+ cells (< 1 x 10(-3) expressing the HLADR-/CD34-, CD33+/HLADR- and CD34+/HLADR- phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117+/CD15+ blast cells and eight had the CD117+ phenotypes detected at low frequencies (< 1 x 10(-3)) in normal BM. In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117+/CD15+ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.

The introduction of imatinib therapy in nonclinical trial CML patients is associated with potentially irreversible acute renal injury, and the long-term treatment may cause a clinically relevant decrease in the estimated GFR.

Aim-To explore the role of phenotypic changes as possible limiting factors in the immunological detection of minimal residual disease in patients with acute myeloid leukaemia (AML). Methods-20 relapses were evaluated, with special attention to changes in the criteria used for the definition of a phenotype as "aberrant". In all cases the same monoclonal antibody and fluorochrome were used at diagnosis and in relapse. Results-Six out of the 16 patients showed aberrant phenotypes at diagnosis. At relapse, no changes in the aberrant phenotypes were detected in most ofthe patients; nevertheless, in two of the four patients with asynchronous antigen expression this aberration disappeared at relapse. At diagnosis in both cases there were already small blast cell subpopulations showing the phenotype of leukaemic cells at relapse. Ten out of the 16 cases analysed showed significant changes in the expression of at least one of the markers analysed. Conclusions-At relapse in AML the "leukaemic phenotypes" usually remained unaltered, while other phenotypic features-not relevant for distinguishing leukaemic blast cells among normal progenitors-changed frequently; however, they were not a major limitation in the immunological detection of minimal residual disease.

The existence of leukemic-associated phenotypes has been suggested to be a valuable tool for the detection of minimal residual disease (MRD) in AML patients, as they would allow to distinguish leukemic blast cells from normal hematopoietic progenitors. The present study was designed to analyze in which proportion of AML patients the immunological detection of MRD is feasible, based on the presence of aberrant phenotypes that allow the distinction of leukemic from normal cells. For this purpose we have prospectively investigated the blast cells from 40 AML patients at diagnosis with a large panel of MoAb in double and triple staining combinations analyzed at flow cytometry, in order to detect aberrant phenotypes on blast cells (lineage infidelity, antigenic overexpression, and asynchronous antigenic expression, as well as aberrant light-scatter pattern). In the analysis of the 40 AML cases more than one blast cell subset, distinguished by its different antigenic expression, was detected in 85% of the patients: five different phenotypic blast cell subsets were observed in six cases, four in 13 patients, three subsets in three cases, and two in 12 patients; only six cases showed a homogeneous phenotypical blast cell population. Twenty-nine of the 40 AML cases analyzed (73%) showed the existence of at least one aberrant phenotype: in 15 cases the myeloid blast cells co-expressed lymphoid-associated antigens (CD2, CD5, CD7, and/or CD19)--lineage infidelity--; asynchronous antigen expression was detected in 25 patients (CD34+CD56+, CD34+CD11b+, CD34+CD14+, CD117+CD15+, CD33-CD13+, CD13-CD15+, HLADR + CD15 , HLADR-CD14+CD11b+ CD4+); seven cases displayed antigen overexpression (CD13, CD33, CD15, or CD14); and in 13 patients leukemic cells had an abnormal FSC/SSC distribution according to their phenotype. These results suggest that immunological methods for the detection of MRD based on the existence of aberrant phenotypes could be used in the majority of AML patients.

The existence of leukemic-associated phenotypes has been suggested to be a valuable tool for the detection of minimal residual disease (MRD) in AML patients, as they would allow to distinguish leukemic blast cells from normal hematopoietic progenitors. The present study was designed to analyze in which proportion of AML patients the immunological detection of MRD is feasible, based on the presence of aberrant phenotypes that allow the distinction of leukemic from normal cells. For this purpose we have prospectively investigated the blast cells from 40 AML patients at diagnosis with a large panel of MoAb in double and triple staining combinations analyzed at flow cytometry, in order to detect aberrant phenotypes on blast cells (lineage infidelity, antigenic overexpression, and asynchronous antigenic expression, as well as aberrant light-scatter pattern). In the analysis of the 40 AML cases more than one blast cell subset, distinguished by its different antigenic expression, was detected in 85% of the patients: five different phenotypic blast cell subsets were observed in six cases, four in 13 patients, three subsets in three cases, and two in 12 patients; only six cases showed a homogeneous phenotypical blast cell population. Twenty-nine of the 40 AML cases analyzed (73%) showed the existence of at least one aberrant phenotype: in 15 cases the myeloid blast cells co-expressed lymphoid-associated antigens (CD2, CD5, CD7, and/or CD19)--lineage infidelity--; asynchronous antigen expression was detected in 25 patients (CD34+CD56+, CD34+CD11b+, CD34+CD14+, CD117+CD15+, CD33-CD13+, CD13-CD15+, HLADR + CD15 , HLADR-CD14+CD11b+ CD4+); seven cases displayed antigen overexpression (CD13, CD33, CD15, or CD14); and in 13 patients leukemic cells had an abnormal FSC/SSC distribution according to their phenotype. These results suggest that immunological methods for the detection of MRD based on the existence of aberrant phenotypes could be used in the majority of AML patients.

This study aimed at evaluating the use of immunophenotyping (IMP) in the identification of blast cells in the cerebrospinal fluid (CSF) of children and adolescents with acute lymphoblastic leukemia (ALL). Sixty-seven patients aged 18 years or younger were included. Fifty-five CSF samples were analyzed at initial diagnosis and 17 at the time of relapse. A cytological analysis (CA) was performed in all 72 samples, while IMP was done in 63. Blasts were identified in only three samples by CA, whereas all three samples were found negative by IMP, one of which had no isolation of nucleated cells after centrifugation. Among the samples analyzed by IMP, 11 showed a positive blast count, two of which had been inconclusive using CA. No equivalence was found between CA and IMP results (p = 0.55). CSF IMP positivity was not associated with other risk factors for ALL relapse. Among the 55 patients included at the time of diagnosis of ALL, eight relapsed during follow-up. Considering the cases of central nervous system (CNS) relapse, one of the patients belonged to the CSF IMP-positive group (11%) at diagnosis, and the other two cases, to the IMP-negative (5%) group. Detection of CSF blast cells using IMP was associated with a worse overall (p < 0.0001) and event-free survival (p < 0.0001). These results show that CSF IMP may be a useful additional method to conventional CA in the diagnosis of CNS involvement in ALL, and for the identification of high-risk subgroups that would benefit from an intensified therapy.

Objectives: This study aims at verifying the prevalence of gastroesophageal reflux in asthmatic children, and at determining the sensitivity and specificity of the reflux index for the diagnosis of gastroesophageal reflux disease.Methods: Sixty-nine children, aged 1-5 years, with asthma, were studied by 24-hour pH monitoring. The patients were randomly selected.Results: Ages varied from 12.4 to 63.1 months, mean age = 30.79 months, and 62.3% were males.Gastroesophageal reflux was observed in 68.1% of the children. The patients were divided into two groups, moderate and severe asthma, and gastroesophageal reflux was diagnosed in 58.5 and 82.1% of the cases, respectively. Occult gastroesophageal reflux occurred in 31.8% of the cases. The reflux index showed an sensitivity of 89.4%, specificity of 95.5%, positive predictive value of 97.7% and negative predictive value of 80.8%. Conclusions:The results of this study indicate a relationship between gastroesophageal reflux and asthma, and suggest that the reflux index as a single parameter of pH monitoring has good sensitivity and specificity for the diagnosis of gastroesophageal reflux disease.J Pediatr (Rio J). 2007;83(6):535-540: Asthma, gastroesophageal reflux, child, pH monitoring.