A new automated flow cytometry data analysis approach for the diagnostic screening of neoplastic B-cell disorders in peripheral blood samples with absolute lymphocytosis.

Currently, multiparameter flow cytometry immunophenotyping is the selected method for the differential diagnostic screening between reactive lymphocytosis and neoplastic B-cell chronic lymphoproliferative disorders (B-CLPD). Despite this, current multiparameter flow cytometry data analysis approaches still remain subjective due to the need of experienced personnel for both data analysis and interpretation of the results. In this study, we describe and validate a new automated method based on vector quantization algorithms to analyze multiparameter flow cytometry immunophenotyping data in a series of 307 peripheral blood (PB) samples. Our results show that the automated method of analysis proposed compares well with currently used manual approach and significantly improves semiautomated approaches and, that by using it, a highly efficient discrimination with 100% specificity and 100% sensitivity can be made between normal/reactive PB samples and cases with B-CLPD based on the total B-cell number and/or the sIgkappa+/sIglambda+ B-cell ratio. In addition, the method proved to be able to detect the presence of pathologic neoplastic B-cells even when these are present at low frequencies (<5% of all lymphocytes in the sample) and in poor-quality samples enriched in 'noise' events.

Genetic heterogeneity of BCR/ABL+ adult B-cell precursor acute lymphoblastic leukemia: impact on the clinical, biological and immunophenotypical disease characteristics.

Philadelphia-positive (Ph(+)) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous disease with a very poor prognosis. In this study, we analyzed the frequency of supernumerary Ph, trisomy 8, monosomy 7, and del(9p21) by FISH and its relationship with the characteristics of the disease, in 46 BCR/ABL(+) adult BCP-ALL patients. The frequency of supernumerary Ph, trisomy 8, monosomy 7 and del(9p21) was 30%, 20%, 15%, and 24%, respectively. Although all patients displayed a BII/common phenotype, supernumerary Ph and trisomy 8 were associated with higher expression of CD19 and CD22 and of CD19, CD34, CD45, and HLA-DR, respectively; in turn, cases with monosomy 7 showed lower CD19, CD22, CD34, and cCD79a and del(9p21)(+) blasts were CD13(-) and CD33(-). Overall, similar clinical and hematological features were observed at presentation, independently of the underlying genetic abnormalities. However, relapse-free survival (RFS) was significantly shorter in cases with supernumerary Ph, trisomy 8, and del(9p21), the latter being the most powerful independent prognostic factor for RFS.

Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities.

Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). In the present study, we have explored the incidence of both typical and atypical iFISH patterns of BCR/ABL gene rearrangements in a series of 168 consecutive BCR/ABL+ patients--135 CML, 31 precursor B-ALL and two acute myeloblastic leukemia (AML) cases--and established their underlying genetic alterations through further molecular and chromosome analyses. Two different FISH probes (Vysis Inc., Downers Grove, IL, USA) were used: the LSI BCR/ABL dual color extra signal (ES) and the dual color dual fusion BCR/ABL probe (D-FISH). Our results show that most BCR/ABL+ patients (83%, including 88% of all CML, 61% of ALL and one of two AML) displayed typical iFISH patterns of either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of the two AML) or minor (m) BCR/ABL gene rearrangements (1% of all CML and 48% of ALL cases) with the two probes. Further molecular and cytogenetic studies confirmed the presence of such typical rearrangements in all except one of these ALL cases who had coexistence of an MBCR/ABL and an mBCR/ABL gene rearrangement together with monosomy 9. In the remaining 29 cases (17%), up to five different atypical iFISH patterns were detected with the ES probe. Atypical iFISH patterns were most frequently due to additional numerical changes--most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22 (1%). Deletion of 9q sequences proximal to the breakpoint were also frequently observed with the ES probe (8%). Application of the D-FISH probe showed that in most of these latter cases (5%) deletion of 22q sequences distal to the breakpoint also occurred. The remaining cases with atypical iFISH had cryptic insertion of BCR in 9q34 (1%). Exact interpretation of each iFISH pattern was supported by FISH on metaphases and molecular determination of the BCR breakpoint. In summary, our results indicate that despite the high incidence of typical iFISH patterns of BCR/ABL gene rearrangements, atypical patterns are also found in BCR/ABL+ acute leukemias; the precise definition of the alteration present in individual cases is dependent on metaphase studies and molecular definition of the breakpoint.

Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10, CD34, CD13 and CD38 expresssion.

The Philadelphia chromosome (Ph+) reflects a balanced reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11.2] involving the BCR and ABL genes. At present, detection of BCR/ABL gene rearrangements is mandatory in precursor-B-ALL patients at diagnosis for prognostic stratification and treatment decision. In spite of the clinical impact, no screening method, displaying a high sensitive and specificity, is available for the identification of BCR/ABL+ precursor-B-ALL cases. The aim of the present study was to explore the immunophenotypic characteristics of precursor B-ALL cases displaying BCR/ABL gene rearrangements using multiple stainings analyzed by quantitative flow cytometry in order to rapidly (<1 h) identify unique phenotypes associated with this translocation. From the 82 precursor-B-ALL cases included in the study 12 displayed BCR/ABL gene rearragements, all corresponding to adult patients, four of which also displayed DNA aneuploidy. Our results show that BCR/ABL+ precursor B-ALL cases constantly displayed a homogeneous expression of CD10 and CD34 but low and relatively heterogeneous CD38 expression, together with an aberrant reactivity for CD13. In contrast, this unique phenotype was only detected in three out of 70 BCR/ABL cases. Therefore, the combined use of staining patterns for CD34, CD38 and CD13 expression within CD10-positive blast cells is highly suggestive of BCR/ABL gene rearrangements in adults with precursor B-ALL.

Detection of the Mbcr/abl translocation in chronic myeloid leukemia by fluorescence in situ hybridization: comparison with conventional cytogenetics and implications for minimal residual disease detection.

The correlation between the detection of the Philadelphia chromosome by conventional cytogenetics and the identification of Mbcr/abl translocation by fluorescence in situ hybridization (FISH) in both metaphase and interphase cells is prospectively analyzed in a group of 21 chronic myeloid leukemia (CML) patients. To gain insight into the sensitivity and specificity of the detection of the bcr/abl translocation by FISH, a group of 10 healthy volunteers was also studied. Our results show that for the detection of bcr/abl translocation in CML patients, FISH is more sensitive than conventional cytogenetics because it detects significantly higher proportions of cells carrying the translocation both in metaphase (P < .0002) and interphase nuclei (P < .003). Moreover, in the metaphases of the controls analyzed, no bcr/abl+ chromosome was detected that makes the colocalization of bcr and abl signals in the CML patients highly specific. Conversely, in control interphase nuclei, a small proportion of cells (ranging between 0% and 3%, mean value of 1.7% +/- 0.9%) displaying colocalization of both signals is usually detected. This limits, at least for the moment, the routine use of FISH for the detection of minimal residual disease in CML patients at levels lower than 10(-1).

Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3% of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.

Positive response to imatinib mesylate therapy for childhood chronic myeloid leukemia

Chronic myeloid leukemia (CML) is rare in the pediatric population, accounting for 2-3 percent of childhood leukemia cases, with an annual incidence of one case per million children. The low toxicity profile of imatinib mesylate has led to its approval as a front-line therapy in children for whom interferon treatment has failed or who have relapsed after allogeneic transplantation. We describe the positive responses of 2 children (case 1 - from a 7-year-old male since May 2005; case 2 - from a 5-year-old female since June 2006) with Philadelphia-positive chromosome CML treated with imatinib (300 mg/day, orally) for up to 28 months, as evaluated by morphological, cytogenetic, and molecular approaches. Our patients are alive, are in the chronic phase, and are in continuous morphological complete remission.

Changes in both the membrane and the enzyme content of individual zymogen granules are associated with sodium taurocholate-induced pancreatitis in rats.

1. After monitoring the changes associated with necrotizing acute pancreatitis in rats from early stages to 24 h after infusion of 5% sodium taurocholate in the choledocus, we characterized by flow cytometry the zymogen granules that still remained in the pancreas 18 h after sodium taurocholate infusion in order to explore whether alterations in the enzyme content and/or in the composition of the granule membrane could be related to the intracellular mechanisms involved in the development of necrotizing acute pancreatitis. 2. Significant increases in the haematocrit, plasma and peritoneal exudate amylase levels and oedema were observed from the third hour after 5% sodium taurocholate infusion onwards. Additionally, cell alterations such as hypergranulation, dilatation of the endoplasmic reticulum and autophagic vacuoles were found 3 and 6 h after infusion. DNA decrease, degranulation and necrosis were observed from 12 h after sodium taurocholate infusion onwards. 3. Flow cytometric measurements of zymogen granules isolated from rat pancreas 18 h after 5% sodium taurocholate infusion revealed a significant decrease in their internal complexity without major changes in their size. Double staining of granules with Tetragonolobus purpureus lectin, which specifically binds L-fucose and specific anti-trypsinogen or anti-amylase antisera, showed that rats with induced pancreatitis have decreased amounts of L-fucose in the membrane glycoconjugates and lower enzyme content (70% and 30% less for trypsinogen and amylase respectively). 4. A decrease in L-fucose in the membrane together with membrane abnormalities observed by electron microscopy in zymogen granules isolated 18 h after sodium taurocholate infusion indicate an altered synthesis of new granules or lysis of preformed zymogen granules which would favour differential loss of granular enzymes, mainly trypsinogen, which in turn could increase the severity of disease.

[Plasminogen deficiencies in 2 Spanish families. Response to the administration of DDAVP]. / Deficiencias de plasminógeno en dos familias españolas. Respuesta a la administración de DDAVP.

We describe two families with heterozygous plasminogen deficiency. In the first the patient was a 27 year-old female who suffered an acute episode of ischemic cerebrovascular disease affecting the left temporal lobe documented by arteriographic, gammagraphic and CAT studies. She had no family history of thrombotic conditions. In the other family the propositus was a 31 year-old man with spontaneous deep venous thrombosis in the left leg. His father was also symptomatic, with a history of recurrent thrombotic complications after predisposing factors, that included multiple venous thrombosis and a pulmonary embolism. Laboratory data showed normal hemostasis test results. Antigenic and functional levels of protein C, protein S and antithrombin III were within normal limits. The only abnormality found was decreased plasminogen activity in plasma; antigenic and functional levels were reduced to about half-normal levels. In both cases crossed immunoelectrophoresis revealed a normal migration pattern of plasminogen. Thus, we conclude that our patients were carriers of congenital hypoplasminogenemia or familial type I plasminogen deficiency, due to decreased synthesis. We also reported on fibrinolytic response to infusion of DDAVP, a synthetic analogue of the antidiuretic hormone. Fibrinolytic activity was normal in basal conditions as well as in response to DDAVP infusion.

Natural anticoagulant proteins and antiphospholipid antibodies in systemic lupus erythematosus.

OBJECTIVE: Thrombosis is a relatively common complication in patients with systemic lupus erythematosus (SLE) and is strongly associated with the presence of antiphospholipid antibodies (aPL). The mechanism involved in the pathogenesis of this prothrombotic state remains obscure. We studied 4 natural anticoagulant proteins: protein C, protein S, antithrombin III, and plasminogen in 50 patients diagnosed with SLE. METHODS: Protein C, antithrombin III, and plasminogen were measured by chromogenic substrates and total and free protein S by electrophoresis. We also determined the prevalence of different aPL (lupus anticoagulant and antibodies against cardiolipin, phosphatidylserine, and phosphatidylinositol). RESULTS: Ten patients (20%) had a history of thrombosis. Some type of aPL was present in 26 patients (52%). Nine of the 10 patients with history of thrombosis had aPL (p = 0.007). Functional assays for protein C, antithrombin III, and plasminogen were in the normal range in all patients. Low free protein S levels were documented in 19 patients and were associated with the presence of aPL (13/19 were aPL positive) (p < 0.05). Only 4 patients with acquired free protein S deficiency had a history of thrombosis. CONCLUSION: This study shows an association between aPL and reduced free protein S levels in patients with SLE. Further studies are needed to determine the mechanism and role of this acquired deficiency in the pathogenesis of thrombotic episodes in patients with SLE.

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages.

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.

Incidence and clinical characteristics of hereditary disorders associated with venous thrombosis.

At present, different congenital defects in several proteins--antithrombin III (AT III), protein C (PC), protein S (PS), and plasminogen (PLG)--are known to be causes of hereditary predisposition to thrombosis (thrombophilia). The incidence of these hereditary disorders in our 204 patients (106 males and 98 females) with venous thromboembolism were 4% (three cases deficient in PC, three in PS, two in PLG, and one patient in AT III). Their families were studied. In all cases the disorders were inherited as an autosomal dominant trait. The first thrombotic episodes occurred at a age of below 40 years. There was no relationship between protein levels and the occurrence of thrombosis, although a significant relationship was observed between a positive history of thromboembolic disease and a diagnosis of protein deficiencies. We evaluated the differences between primary thrombosis and secondary thrombosis. The most common thrombotic sites were the deep veins. There were no differences between males and females. Evaluation of PC, PS, AT III, and PLG in patients with thromboembolic disease should be considered.

Clinical, biological, and immunophenotypical characteristics of B-cell chronic lymphocytic leukemia with trisomy 12 by fluorescence in situ hybridization.

The clinical, biological, and immunophenotypical characteristics of B-cell chronic lymphocytic leukemia (B-CLL) patients with trisomy 12 detected by fluorescence in situ hybridization (FISH) using a chromosome 12 alpha-centromeric probe (D12Z3) were analyzed in the present study. From a total of 104 consecutive B-CLL patients, 21 (20%) displayed trisomy 12, the percentage of trisomic cells ranging from 13% to 76%. From the clinico-biological point of view, patients with trisomy 12 were associated with atypical CLL morphology (43% vs 10%, P = 0.04) and BM diffuse pattern (75% vs. 25%, P = 0.02) together with increased WBC counts (141 +/- 220 vs. 58 +/- 67 x 10(9)/L, P = 0.04). In contrast, no association was detected between the presence of trisomy 12 and other disease characteristics such as age, sex, clinical stage, hepatomegaly, lymphadenopathies, haemoglobin levels and platelet counts, and the cell cycle distribution of PB leukocytes in both groups of patients. Trisomy 12 patients had a significantly higher expression of the FMC7 antigen both in percentage (34 +/- 34% vs. 13 +/- 20%, P = 0.02) and absolute numbers (29 +/- 62 vs. 7 +/- 17 x 10(9)/L, P = 0.007). No major differences were found regarding the expression of mouse rosettes, CD19+, and CD19+/CD5+ lymphocytes. Upon analyzing the correlations between the disease characteristics of trisomy 12 cases, significant associations were found between the percentage of trisomic cells and both the WBC count (r = 0.52, P = 0.02) and the PB lymphocyte count (r = 0.60, P = 0.007).(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and biological characteristics of myelodysplastic syndromes with nulisomy Y by fish.

BACKGROUND AND OBJECTIVE: In the present study we analyzed the incidence of nulisomy Y by fluorescence in situ hybridization in a group of 24 males diagnosed with myelodysplastic syndromes (MDS). We explored the relationship between this chromosome abnormality and other clinical and biological disease characteristics. METHODS: Loss of chromosome Y was present in 7 out of the 24 males analyzed (29%); the number of cells carrying this chromosome aberration ranged between 19% and 90%. From the clinicobiological point of view, the group of patients with nulisomy Y showed a higher incidence of RA and RAS FAB subtypes (p = 0.04), a lower WBC count (p = 0.04), a lower proportion of blast cells both in PB (p = 0.009) and BM (p = 0.06) associated with a decreased myeloid/erythroid ratio (p = 0.01). RESULTS: No clear association was detected between loss of chromosome Y and other numerical chromosome abnormalities involving chromosomes 7 and 8. In contrast, 2 out of the 7 cases with loss of chromosome Y also displayed monosomy 1 by FISH. However, the use of appropriate dual stainings showed that these two abnormalities were present in different cell populations (that is, they never coexisted in the same cell population), which supports the notion of the existence of clonal heterogeneity in MDS patients. INTERPRETATION AND CONCLUSIONS: From the prognostic point of view, MDS patients with loss of chromosome Y displayed a higher survival rate, although these differences did not reach statistical significance.

[Long-term culture: evidence of the capacity of stroma to sustain hematopoiesis of a second inoculum]. / Cultivo a largo plazo: evidencia de la capacidad del estroma para sustentar la hematopoyesis de un segundo inóculo.

PURPOSE: To analyze, in two-stages long term bone marrow cultures (LTBMC), the efficiency of the method used for irradiation of the stroma and to check if after that, it's capable to support the hematopoiesis. MATERIAL AND METHODS: We have used for the first inoculum, bone marrow cells from eight controls. They were cultured at 2 x 10(6) cells/ml concentration until obtaining a fully confluent stroma which was irradiated with 15 Gy. After that, over this layer, we put a second inoculum with bone marrow cells in four cases, and with peripheral blood cells in four others, at a concentration of 1 x 10(5) cells/mL. In four cases the first inoculum was from a man and the second from a woman. The culture's haematopoietic activity was determined by the number of CFU-GM, total cell counts and the differential counts during four weeks. At the end of the study we recovered the cells from the supernatant of the culture and they were analyzed by in situ hybridization with an alpha centromeric probe specific to the X chromosome. RESULTS: In all cases we obtained a confluent stroma with production of haematopoiesis (cobblestone areas). The cells recovered at the end of the culture were, in all cases, from the second inoculum because they had a double signal with the X probe. The number of CFU-GM obtained was higher in bone marrow than in peripheral blood (34.5 and 9.2 respectively) however, the total cells were similar in both cases (2.3 x 10(5) and 2.9 x 10(5)) although the cellular subtypes varied depending on the second inoculum (B.M. or P.B.). CONCLUSION: Our data confirm that the dose of 15 Gy eradicate the haemopoiesis of first inoculum, which allowed to analyze stroma and progenitor cells, separately. In addition, the stroma is capable to support the hematopoiesis generated by progenitor cells from peripheral and bone marrow.

Continuous oral cytarabine ocfosfate with interferon-alpha-2b for patients with newly diagnosed chronic myeloid leukaemia: a pilot study.

Recombinant(R) interferon alpha (r-IFN-alpha) has been shown to be an effective drug for chronic myeloid leukaemia (CML). However, higher response rates can be achieved using cytarabine along with r-IFN-alpha. YNK01 is a derivative of cytosine arabinoside for oral administration. So far, the only published experience with continuous YNK01 was in advanced CML (10 cases). We have performed a pilot study to evaluate the efficacy and toxicity of the combined therapy r-IFN-alpha and daily oral YNK01 in patients with newly diagnosed Ph+ CML. Ten previously untreated patients were included in the study. Among those patients evaluable for cytogenetic response, 87% (seven out of eight) reached a major cytogenetic response with four reaching complete cytogenetic response (50%). The most significant side-effects were gastrointestinal. Macrocytic anaemia was observed in three patients. In conclusion, continuous oral administration of YNK01 in combination with IFN-alpha is safe and can result in high-cytogenetic response rates.

Gains of chromosome 22 by fluorescence in situ hybridization in the context of an hyperdiploid karyotype are associated with aggressive clinical features in meningioma patients.

BACKGROUND: Meningiomas usually are considered to be benign tumors; however, 10-20% of cases recur. Few disease characteristics have proved to have prognostic impact for predicting disease free survival. The objective of the current study was to explore the prognostic value of numeric abnormalities of chromosome 22 for meningioma patients. METHODS: In this study, the authors prospectively analyzed the incidence of numeric chromosome abnormalities of chromosome 22 by interphase fluorescence in situ hybridization, using a specific probe for the bcr gene located in chromosome 22q11.2, on a total of 88 consecutive meningioma patients. The authors also analyzed its correlation with both the clinicobiologic characteristics at presentation and the patient's outcome. RESULTS: The authors' results show that monosomy 22 was present in 49% of the cases and that this numeric chromosomal abnormality is not associated with other prognostic features of the disease. In contrast, gains (trisomy/tetrasomy) of chromosome 22 were detected in 8 (9%) cases who simultaneously showed gains for other chromosomes and represent an adverse prognostic factor regarding disease free survival (P = 0.001); in addition, trisomy/tetrasomy 22 was more frequently related to younger patients (P = 0.001), aggressive histopathologic features (P < 0.000), a greater incidence of DNA aneuploidy (P =0.006), and a higher proportion of S-phase tumor cells (P = 0.02). CONCLUSIONS: In summary, the authors conclude that loss of a copy of chromosome 22 is a frequent finding in meningioma tumors, but it does not affect the clinical outcome of these patients. In contrast, gains (trisomy/tetrasomy) of chromosome 22, in the context of an hyperdiploid karyotype, although much less frequent, are associated with a more aggressive disease course.

Association between trisomy 8 and the immunophenotype of blast cells from acute leukemias secondary to a myelodysplastic syndrome or chronic myeloproliferative disorders.

In the present study we have used FISH to analyze the incidence of trisomy 8 in acute leukemias following either a primary myeloproliferative disorder (MPD) or a myelodysplastic syndrome (MDS) and correlated it with both the immunophenotype and the cell-cycle distribution of the leukemic blast cells. Six of the 21 (28%) acute leukemias studied displayed trisomy 8 by FISH. The number of trisomic cells in these cases ranged from 20 to 84%, with a mean of 46 +/- 24%. Trisomy 8 was associated with a homogeneous population of leukemic cells, phenotypically characterized by CD34+/HLADR+/CD13+/CD33+/CD11b-/ CD15-/CD14-. No significant differences were observed on the proliferative rate of cases with trisomy 8, as compared with blast cells from the remaining patients. Overall, our findings suggest that in acute leukemias secondary to MPD or MDS, trisomy 8 is associated with a blockade of myeloid maturation at an early step of the differentiation process.

The flow cytometric pattern of CD34, CD15 and CD13 expression in acute myeloblastic leukemia is highly characteristic of the presence of PML-RARalpha gene rearrangements.

BACKGROUND AND OBJECTIVE: Rapid identification of AML patients carrying the t(15;17) translocation for treatment decision-making is currently made on the basis of morphologic screening. However, the existence of both false positives and negatives highlights the need for more objective methods of screening AML cases and further molecular confirmation of the t(15;17) translocation. DESIGN AND METHODS: In the present study we analyzed a total of 111 AML cases in order to investigate whether immunophenotyping based on the assessment of multiple-stainings analyzed at flow cytometry could improve the sensitivity and specificity of morphologic identification of acute promyelocytic leukemia (APL) carrying the t(15;17) translocation. FISH analysis was used as a complementary technique for cases in which morphology and molecular biology yielded discrepant results. RESULTS: Concordant results between morphology and RT-PCR were found in 102/111 (91.8%) cases: 34 patients had M3/PML-RARalpha+ and 68 non-M3/PML-RARalpha- disease. Nine cases showed discrepants results. Multivariate analysis showed that the best combination of immunologic markers for discriminating between M3/PML-RARalpha+ and non-M3/PML-RARalpha- cases was that of the presence of heterogeneous expression of CD13, the existence of a single major blast cell population, and a characteristic CD34/CD15 phenotypic pattern (p<0.02). A score system based on these parameters was designed, and the 34 M3/PML-RARalpha+ cases showed a score of 3 (presence of the 3 phenotypic characteristics). In contrast, only 1 out of the 68 (1.3%) non-M3/PML-RARalpha- cases had this score, most o these latter cases (53/68, 78%) scoring either 0 or 1. Therefore, among these cases, immunophenotyping showed a sensitivity of 100% and a specificity of 99% for predicting PML/RARalpha gene rearrangements. Of the 9 cases in which morphology and molecular biology results were discrepant, four cases displayed M3 morphology without PML/RARalpha rearrangements by RT-PCR. In only one of these 4 cases did the immunophenotype score 3, this being the only FISH positive case. From the remaining five discrepant cases (non-M3 morphology while positive for PML/RARalpha) two cases had a phenotypic score of 3 and were FISH positive while the other three were negative by FISH. Upon repeating RT-PCR studies, two of these latter three cases became negative. INTERPRETATION AND CONCLUSIONS: Our results show that immunophenotyping may be of great value for quick screening of APL with PML/RARalpha rearrangements.