Mario Masi

Incidence of chromosome abnormalities and clinical significance of karyotype in de novo acute myeloid leukemia

Cytogenetic studies with high-resolution banding were performed on specimens from 132 consecutive patients with de novo acute myeloid leukemia (AML). All patients were treated according to therapeutic protocols in the same institution. Clonal abnormalities were detected in 97 of the 124 patients in whom an adequate number of mitoses was obtained (78.2%). Neither sex, FAB classification, WBC, or the extent of bone marrow infiltrate affected the rate of chromosomal aberrations, whereas patients younger than 40 years had a greater proportion of normal karyotypes (p = 0.047). Two different chromosomal classifications were evaluated: the presence of normal and abnormal metaphases (NN-AN-AA classification), and a classification in cytogenetic categories, the latter being based on the frequency of cytogenetic abnormalities. Both classifications were found to correlate significantly with the clinical outcome. They also showed independent prognostic significance when age, sex, and FAB morphology were considered in a multivariate analysis. Two abnormalities were closely associated with specific clinical-pathologic subsets of AML. All the 15 patients with t(15;17) had acute promyelocytic leukemia; this translocation was not found in any other subset of AML. Eight of the nine patients presenting rearrangements at 11q23 belonged to a FAB subset with monocytic differentiation (M4 and M5). Our data suggest that cytogenetic findings should influence the therapeutic approach to AML. In particular, young patients with karyotypes associated with poor responses may be considered for more eradicating treatments, including allogenic bone marrow transplantation.

Intensive Treatment of Patients Age 60 Years and Older with De Novo Acute Myeloid Leukemia Analysis of Prognostic Factors

This study aimed to define pre‐treatment parameters with prognostic significance in elderly patients with de novo acute myeloid leukemia (AML) who were treated with aggressive regimens.

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)

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

Summary. 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.

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.

Cytogenetic analysis is non-informative for assessing the remission rate in chronic myeloid leukemia (CML) patients on interferon-α (IFN-α) therapy

Abstract Cytogenetic analysis is considered pivotal for assessing the remission rate in CML patients on IFN therapy. On the basis of general agreement, at least 25 metaphases should be analyzed in each case. The main limitations to this approach are: 1) the small number of analyzable metaphases generally found in cytogenetic preparations from IFN-α-treated patients; and 2) the inability of this technique for scoring interphase cells. We compared the results of cytogenetic analysis and double-color FISH detection of bcr/abl genes fusion in 13 CML patients on IFN-α therapy (marrow sampling for cytogenetic and FISH analysis was carried out after 12 months in all patients and repeated after 18 months of IFN therapy in patients 4, 6, and 8). In five specimens, 20 to 25 cells were evaluable for cytogenetic examination, in another five no analyzable metaphases were scored, and in the remaining six samples two to 14 cells could be analyzed. With FISH detection at least 100 cells were easily scored in each specimen (mean number, 175). Comparing the results carried out with the two methods in different samples it emerged that cytogenetic analysis led to improper conclusions as regards the rate of Ph positivity, even in those patients where 20–25 metaphases were analyzed. Although many more cases have to be studied to establish the role of FISH analysis in Ph-positive patients, we are of the opinion that cytogenetic analysis is unfit for easily and accurately assessing the actual quality of remission in IFN-treated subjects.

A metastatic breast carcinoma presenting as autoimmune hemolytic anemia.

The authors describe the case of a 75-year-old female who was hospitalized for anemia of unknown origin. Physical examination revealed a swelling in the right mammary region, where a mastectomy scar was present from surgery for a breast carcinoma. On admission, laboratory tests disclosed anemia (Hb, 8.5 g/dl), with a reticulocyte count of 65,000/mm3 and slightly increased bilirubin. Immunohematologic study revealed the presence of a red cell autoantibody with anti-D specificity in the serum and in the eluate from the patients erythrocytes. A biopsy of the swelling was performed and histologic examination showed the presence of metastatic cells of breast carcinoma. The patient was given chemotherapy and radiotherapy. At this writing the anemia was absent, the Immunohematologic study was negative, the swelling was greatly reduced, and no other metastatic lesions of breast carcinoma were present.

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.