Germano Aronica

Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia

We have analyzed the expression of Tdt and CD7 in 335 cases of unequivocal acute myeloid leukemia (AML). Tdt was expressed in 80 (25%) of 321 evaluable cases. Twenty-six of 77 (34%) Tdt+ patients assessable for response, entered complete remission (CR) vs 121 of 209 (58%) Tdt− cases (P < 0.001). cd7 was expressed in 102 of 332 (30%) evaluable cases; 37 of 93 assessable (40%) cd7+ patients attained a CR as compared to 114/204 (56%) CD7− (P = 0.013). Duration of survival was significantly shorter for patients with CD7+ or Tdt+ AML (P = 0.006 and 0.001, respectively). In a multivariate analysis, Tdt was found to significantly adverse achievement of CR (P = 0.018), while CD7 affected duration of CR (P = 0.037). Overall the expression of either Tdt or CD7 correlated with a relatively high expression of CD34 (P < 0.001), gp-170 (P = 0.003), lymphoid antigens (LyAg) (P < 0.001), t(9;22) or anomalies of chromosome 5/7 (P < 0.001). finally, we pooled the patients into four phenotypic classes, according to the presence of tdt, cd7 or both: [tdt−CD7−], [Tdt+CD7−], [Tdt−CD7+] and [Tdt+CD7+]. The category [Tdt+CD7+] was characterized by a more unfavorable outcome as suggested by a lower rate of CR (P < 0.001) and a shorter duration of survival as compared to cases [tdt−CD7−], [Tdt+CD7−] and [Tdt−CD7+] (P = 0.002). This figure is consistent with the frequent convergence in the subset [Tdt+CD7+] of GP-170 positivity (P = 0.003), translocation t(9;22), anomalies of chromosome 5 and/or 7 (P < 0.001) and signs of lineage infidelity (deviant expression of lymphoid antigens) (P < 0.001). we conclude that the expression of tdt or cd7 is associated with an unfavorable outcome and that the combination of both defines a clinical subset with a poorer prognosis due to the significantly higher association with mdr phenotype, and ‘poor prognostic’ chromosomal abnormalities.

Minimally differentiated acute myeloid leukaemia (AML-MO): cytochemical, immunophenotypic and cytogenetic analysis of 19 cases

Summary. We describe our experience in the identification of 19 cases of AML‐MO categorized among 200 consecutive AML cases. Leukaemic cells from our cases were morphologically marked by agranuler basophilic cytoplasm, finely dispersed chromatin and prominent nucleoli. In two cases heavily vacuolated and monocytoid‐shaped blasts were also observed. Cytochemistry (MPO, SBB, αANAE, αNBE, NASDCAE, AP, PAS) was negative in 14 cases, five cases expressing a very faint cytoplasmic positivity for αNBE (not exceeding 30% of the blasts) and αANAE (not exceeding 41%) which was sodium fluoride resistant. In these five cases other monocytic markers (e.g. CD14) were not in favour of myelomonocytic differentiation. All the cases were anti‐MPO positive at frequency > 10%. Phenotypic analysis also revealed myeloid features with all the patients having at least one myeloid antigen (CD13, CD33, CD15), Tdt was expressed in nine cases and CD7 in six cases. All cases but one were positive for CD34. Cytogenetic analysis, performed in 16 cases, showed no adequate growth in two cases and no consistent abnormality in four; among the remaining 10 cases no consistent abnormality was observed, the most common finding was trisomy 8 (two cases) and 4 (two cases) and aberrations of chromosomes 2, 3, 5, 7, 9, 12 and 21. No cases of (t9;22), Ph chromosome were observed. Interestingly three out of five patients with faint αNBE/αANAE positivity relapsed as typical M4 (one case) or M5a (two cases).

P-glycoprotein and terminal transferase expression identify prognostic subsets within cytogenetic risk classes in acute myeloid leukemia

Clinical and biological features were assessed in 204 consecutive de novo adult acute myeloid leukemia (AML) patients who received intensive chemotherapy regimens. Multiparameter flow cytometric assays both of the multidrug resistance (MDR-1)-associated P-glycoprotein (PGP) using the UIC2 monoclonal antibody (MoAb), and of terminal transferase (TdT) were performed. Cytogenetic findings were obtained from 196 patients with high resolution banding. At onset, UIC2 and TdT positivities were detected in 58.5% and 24% of cases, respectively. There were strict correlations either between UIC2 negativity and FAB M3 or between TdT and FAB M0-M1 (P = 0.001 and < 0.0001, respectively). On the other hand, age was significantly associated with cytogenetic risk classes (P < 0.0001). CD34 positivity was highly correlated with TdT expression (P < 0.0001). Moreover, CD7 and CD11b were significantly represented in UIC2+ subset (P < 0.0001). Rhodamine 123 (Rh 123) efflux was significantly higher in 75 UIC2 positive patients compared to 65 UIC2 negative ones (P < 0.001). As regards to cytogenetics, TdT positivity was strongly related either to t(9;22) or single/associated anomalies of chromosome 7; on the other hand, most or all cases with t(8;21) or t(15;17) were UIC2 or TdT negative, respectively. The rate of first complete remission (CR) differed both between UIC2+ and UIC2- cases and between TdT+ and TdT- ones (40% versus 72%, P < 0.001; and 36% versus 61%, P = 0.001, respectively). The survival rates (Kaplan-Meier method) were significantly shorter either in UIC2+ or in TdT+ patients (P = 0.005 and = 0.011, respectively). UIC2 and TdT negative cases showed longer remission duration (P = 0.03 and = 0.22, respectively). The additional effect of UIC2 and TdT on prognosis allowed us to identify two subsets of patients, the first [UIC2- TdT-] at better and the second [UIC2+ TdT+] at worse clinical outcome compared to single UIC2 and TdT cases, concerning CR (P < 0.001), survival (P < 0.0001) and CR duration (P = 0.007). The combinations [UIC2+ TdT-] and [UIC2- TdT+] showed an intermediate clinical course. A strong difference was found between poor risk and intermediate/favorable risk cytogenetic classes with regard to CR rate (P < 0.0001), overall survival and CR duration (P < 0.001). Nevertheless, within the poor risk class, UIC2 positivity was able to identify patients at worst prognosis with regard to CR (P = 0.005), survival (P = 0.02) and CR duration (P = 0.015). On the other hand, UIC2 and TdT negativity allowed us to distinguish patients with longer survival (P = 0.012 and = 0.04, respectively) and CR duration (P = 0.04 and = 0.025, respectively) within the intermediate/favorable risk class. The independent prognostic value of UIC2, TdT and cytogenetic risk classes was confirmed in multivariate analysis. These results suggest that PGP and TdT expressions, together with cytogenetic findings, may represent a basic predictor of chemotherapeutic failure in AML.

CD7 Expression in Acute Myeloid Leukemia

The clinical significance of the expression of CD7 antigen on the blasts of 207 consecutive patients with de novo acute myeloid leukemia (AML) was evaluated. For this purpose, fifty-three CD7+ patients (23 females and 30 males; mean age 52 years) were analyzed and classified into the following subtypes according to French-American-British (FAB) classification: 7 M0, 13 M1, 9 M2, 1 M3, 9 M4, 14 M5. Immunophenotypic studies were carried out by flow cytometry and blast cells were selected on the basis of forward light scatter gating and pan-myeloid marker, either CD13 or CD33. All the CD7+ patients were negative for surface CD3 and T-cell-receptor (TCR) molecules. We found no correlation between CD7 expression and sex, age, hepatosplenomegaly and/or central nervous system involvement. The immaturity of CD7+ leukemic cells was supported by the high expression of CD34 (P = 0.001). CD7 positivity was significantly associated with a white blood cell count (WBC) greater than 100 x 10(9)/L (P = 0.003). P-Glycoprotein (P-170) expression was also evaluated in 135 patients by a flow-cytometric assay: there was a close relationship between CD7 and P-170 positivity (P < 0.001). For remission induction, all patients received therapeutic regimens routinely used for AML. The complete remission (CR) rate was significantly lower in CD7+ cases (32% vs 74%, P = 0.001). The overall survival and disease free survival rate of CD7+ AML was lower than those of CD7- patients (P < 0.001 and = 0.002, respectively). CD7+ AML with coexpression of CD14 had a particularly unfavourable response and prognosis in comparison with CD7+ patients without CD14.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro down-regulation of bcl-2 expression by all-trans retinoic acid in AML blasts

Using flow cytometry, we have investigated the effects of 0.5 μM all-trans–retinoic acid (ATRA) on bcl-2 expression in the blast cells of 25 acute myeloblastic leukemia (AML) patients and the HL-60 cell line after incubation for 6 days. We observed a significant decrease of bcl-2 expression after treatment with ATRA in 12 of 25 AML samples and the HL-60 cells. The mean fluorescence intensity (MFI) ratio for the bcl-2 levels of the ATRA responders (n=12) was reduced to 7.9±4.8 following incubation with ATRA compared with 10.9±6.5 (mean±SD) for control samples incubated without ATRA (p=0.011). There was no significant difference between the baseline bcl-2 MFI ratio in the ATRA responders (11.14±7, n=12) and the non responders (14.18±11.3, n=13;p=0.432). The down-regulation of bcl-2 expression by ATRA was not significantly associated with CD34-negative or -positive AML. There was no correlation between AML subtypes and regulation of bcl-2 expression by ATRA. Complete remission and overall survival were not significantly improved in bcl-2 down-regulated cases. Our data confirm that ATRA can down-regulate the bcl-2 expression in AML blasts. Because many chemotherapeutic agents also operate through the activation of programmed cell death and bcl-2 levels are positively associated with resistance to apoptosis, ATRA can be used in combination chemotherapy to increase the chemosensitivity of some patients with AML.

P-glycoprotein expression in de novo acute myeloid leukemia

Detection of the multidrug resistance P-glycoprotein (PGP) phenotype was performed at the time of diagnosis in 223 patients with acute myeloid leukemia (AML) by flow cytometry using C219 Monoclonal Antibody (MoAb). On the other hand, JSB1 MoAb was tested in 173 of these samples. At onset, PGP was detected in 57.4% of cases with C219 and 75.9% of cases with JSB1. There was no correlation between PGP expression and sex, age, marrow blast percentage or extramedullary disease. On the contrary, strict correlations were noted either between C219 negativity and FAB M3 subtype or between C219 positivity and FAB M5 group (P = 0.003). Significant correlation was found between PGP phenotype and CD7, as 143 of 223 samples had similar patterns of staining with C219 (P < 0.0001). Finally, there was a close relationship between C219 and JSB1 positivity: all the C219+ cases were positive for JSB1 (P < 0.0001). Concerning the karyotype, most patients with monosomy or del (7) were MDR positive; on the other hand, most patients with t(8;21) or t(15;17) were MDR negative. Rh123 accumulation studies showed a significant decrease of mean fluorescence intensities both in C219 and in JSB1 positive cases in comparison with PGP negative ones (P < 0.001). A significant decrease of remission induction rates (CR) was highlighted both between C219+ and C219- and between JSB1+ and JSB1- cases (32.1% v 62.1% and 32.6% v 73.8%, respectively, with P < 0.0001). The overall survival and the remission duration (CCR) were significantly shorter both in C219+ and in JSB1+ patients with no relationship to age. Furthermore, a higher rate of early relapses was noted among MDR+ when compared with MDR- patients both for C219+ and JSB1+ cases. The combination (C219- JSB1+) identified a subset of patients with an intermediate prognosis. On multivariate analysis, C219 and JSB1 were confirmed to be independent prognostic factors for achievement of CR, overall survival and CCR. In conclusion, the assessment of MDR phenotype by flow cytometry is a crucial prognostic factor of treatment outcome in AML.

High-dose chemotherapy in adult acute myeloid leukemia: rationale and results.

Preclinical studies and retrospective evaluations of clinical trials of a number of cytotoxic drugs have provided a rationale for the use of high doses of chemotherapy in adults with acute myeloid leukemia (AML). To maximize cure and remission rates at an acceptable cost in toxicity, many schedules and combinations of dose-intensive chemotherapy have been tested in recent years in patients with de novo disease, cytosine arabinoside (Ara-C) being the most extensively evaluated drug. In this article we review the principal results of both randomized and non-controlled studies. Our analysis indicates that high-dose Ara-C (HIDAC) used during induction results is no substantial benefit relative to conventional doses of drug. On the other hand, consolidation with HIDAC is a major advance in the treatment of this disease. In fact, in individuals less than 60 years of age and a favorable or intermediate-risk karyotype, HIDAC-based regimens have resulted in survival estimates comparable to those of autologous or allogeneic bone marrow transplantation. Yet, the role of HIDAC is irrelevant in younger individuals with an unfavorable cytogenetic pattern and detrimental in patients greater than 60 years of age. Since recently new cytotoxic agents have expanded the armamentarium of antileukemic drugs, well conducted randomized trials of dose intensive chemotherapy still need to be performed to optimize schedules and combinations of drugs in patients with AML.

Automated Haematology Analysers in Acute and Chronic Leukaemias

Accessible online at: http://BioMedNet.com/karger Recently, van der Meer et al. [1] reported a study concerning the potential capability of the Sysmex NE-8000 to detect and characterise the acute or chronic form of both the lymphatic and myeloid leukaemias. For this purpose, they based their results on the use of the combination of the different classification types of the scattergrams and histograms. However, acute myeloid leukaemias could not be distinguished by this approach. Based on our experience [2, 3] we will focus the discussion on four points. (1) Van der Meer et al. [1] excluded 22% of the cases (26/118 patients) with various types of leukaemia because fewer than four out of five technologists agreed upon the classification of the scattergrams and/or histograms. Firstly, it was not specified whether in these cases the blast population extended through both lymphocytic and monocytic/ granulocytic regions or a discrete population was localised to an intermediate or overlapping position. We think it would be useful for these cases to give some morphological and cytochemical information because the scattergrams and histograms broadly reflect the morphology of leukaemic cells (i.e. lymphoblasts near the lymphocytic region, myeloblasts near the monocytic and/or granulocytic regions). Secondly, a percentage of circulating blasts near 50% among other normal cells could generate atypical scattergrams and/or histograms thus hampering a clear identification of leukaemias. (2) The combination of the scattergram type 3 with the histogram type 3 or 5 were observed in all cases of AML-M4 and AMLM5 [1]. It is surprising that the blast cells, especially in AML-M5, were not located in the monocytic region with a scattergram type 2 instead of type 3. We think that the efficiency of the Sysmex NE-8000 at detecting the monocyte population is still a subject of debate. In fact, several authors reported discrepancies between monocyte count as provided manually or by Sysmex NE-8000 and other automated analysers [4, 5]. In this respect, we have previously demonstrated that Coulter VCS allowed a preliminary distinction between immature myeloblastic and monocytic forms, on one hand, and granular and promyelocytic ones on the other [2]. (3) The authors have stated that the combination of the scattergram 2 or 3 with one of the histograms 2, 3 or 4 is very specific for AML (specificity 95%) [1]. Actually, the specificity was very high, but the authors omitted that 14 AML cases (2 M1, 5 M2, 4 M4 and 3 M5) did not show that appearance [table 4; 1]. In fact, the sensitivity was very low (sensitivity 20/20 + 14 = 58%). (4) Finally, van der Meer et al. [1] reported in their discussion that NE-8000 by classifying scattergrams and histograms was able to recognise AML in the majority of the cases and thus to distinguish it from CML. Data concerning CML were not mentioned in the results section; only their table 3 showed the different type of scattergrams and histograms [1]. In an attempt to establish whether there was a possible specific combination for CML, we revised their 17 CML cases and observed that 15 of them appeared with the association of the scattergram type 3 with the histogram type 5. The sensitivity and the specificity are satisfactory (88 and 83%, respectively), but 13 AML cases (2 M1, 4 M2, 4 M4 and 3 M5) showed this combination, thus influencing the predictive value of a positive test (15/28 = 53%; table 1).