Loredana Elia

Dasatinib as first-line treatment for adult patients with Philadelphia chromosome-positive acute lymphoblastic leukemia

Dasatinib is a potent BCR-ABL inhibitor effective in chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia (ALL) resistant/intolerant to imatinib. In the GIMEMA LAL1205 protocol, patients with newly diagnosed Ph(+) ALL older than 18 years (with no upper age limit) received dasatinib induction therapy for 84 days combined with steroids for the first 32 days and intrathecal chemotherapy. Postremission therapy was free. Fifty-three patients were evaluable (median age, 53.6 years). All patients achieved a complete hematologic remission (CHR), 49 (92.5%) at day 22. At this time point, 10 patients achieved a BCR-ABL reduction to < 10(-3). At 20 months, the overall survival was 69.2% and disease-free survival was 51.1%. A significant difference in DFS was observed between patients who showed at day 22 a decrease in BCR-ABL levels to < 10(-3) compared with patients who never reached these levels during induction. In multivariate analysis, BCR-ABL levels of < 10(-3) at day 85 correlated with disease-free survival. No deaths or relapses occurred during induction. Twenty-three patients relapsed after completing induction. A T315I mutation was detected in 12 of 17 relapsed cases. Treatment was well tolerated; only 4 patients discontinued therapy during the last phase of the induction when already in CHR. In adult Ph(+) ALL, induction treatment with dasatinib plus steroids is associated with a CHR in virtually all patients, irrespective of age, good compliance, no deaths, and a very rapid debulking of the neoplastic clone.

Flow cytometric study of potential target antigens (CD19, CD20, CD22, CD33) for antibody-based immunotherapy in acute lymphoblastic leukemia: analysis of 552 cases

Monoclonal antibody (MoAb)-based therapies have opened innovative treatment avenues that have impacted on the management of patients with both neoplastic and non-neoplastic hematological diseases. The aim of our study was to evaluate in a large series of cases of acute lymphoblastic leukemia (ALL) the expression of specific antigens, CD19, CD20, CD22, and CD33, for which MoAbs are available for clinical use. For each antigen, evaluation was based on the percentage of positive leukemic cells and the degree of antigen expression by mean fluorescence intensity (MFI) and antibody binding capacity (ABC) that were correlated with age, immunophenotype, and presence/absence of particular molecular markers. We can document that some of the analyzed antigens showed a degree of expression related to the B-cell maturation profile, and that the antigen expression intensity appeared to vary according to the presence of specific genetic markers. These findings suggest that the possible clinical use of a given MoAb in patients with ALL should take into account both the maturation profile of the leukemic cells and the presence of a given molecular transcript. Only clinical studies will conclusively demonstrate whether the differences in antigenic expression truly correlate with the different therapeutic efficacies of the various clinical grade MoAbs.

Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis

Background In patients with Philadelphia-positive acute lymphoblastic leukemia, resistance to treatment with tyrosine kinase inhibitors is frequent and most often associated with the development of point mutations in the BCR-ABL kinase domain. We aimed to assess: (i) in how many patients BCR-ABL kinase domain mutations are already detectable at relatively low levels at the time of diagnosis, and (ii) whether mutation detection correlates with subsequent response to therapy. Design and Methods We retrospectively analyzed samples collected at diagnosis from 15 patients with Philadelphia-positive acute lymphoblastic leukemia who subsequently received tyrosine kinase inhibitor therapy (dasatinib) by cloning the BCR-ABL kinase domain in a bacterial vector and sequencing 200 independent clones per sample. Results Mutations at relatively low levels (2–4 clones out of 200) could be detected in all patients – eight who relapsed and seven who achieved persistent remission. Each patient had evidence of two to eight different mutations, the majority of which have never been reported in association with resistance to tyrosine kinase inhibitors. In two patients out of six who relapsed because of a mutation, the mutation (a T315I) was already detectable in a few clones at the time of diagnosis. On the other hand, a patient who was found to harbor an F317L mutation is in persistent remission on dasatinib. Conclusions Our results suggest that the BCR-ABL kinase domain is prone to randomly accumulate point mutations in Philadelphia-positive acute lymphoblastic leukemia, although the presence of these mutations in a relatively small leukemic subclone does not always preclude a primary response to tyrosine kinase inhibitors.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party

Background Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Design and Methods The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. Results PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. Conclusions PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Gene expression profiling identifies a subset of adult T-cell acute lymphoblastic leukemia with myeloid-like gene features and over-expression of miR-223

Background Until recently, few molecular aberrations were recognized in acute lymphoblastic leukemia of T-cell origin; novel lesions have recently been identified and a certain degree of overlap between acute myeloid leukemia and T-cell acute lymphoblastic leukemia has been suggested. To identify novel T-cell acute lymphoblastic leukemia entities, gene expression profiling was performed and clinico-biological features were studied. Design and Methods Sixty-nine untreated adults with T-cell acute lymphoblastic leukemia were evaluated by oligonucleotide arrays: unsupervised and supervised analyses were performed. The up-regulation of myeloid genes and miR-223 expression were validated by quantitative polymerase chain reaction analysis. Results Using unsupervised clustering, we identified five subgroups. Of these, one branch included seven patients whose gene expression profile resembled that of acute myeloid leukemia. These cases were characterized by over-expression of a large set of myeloid-related genes for surface antigens, transcription factors and granule proteins. Real-time quantitative polymerase chain reaction analysis confirmed over-expression of MPO, CEBPA, CEBPB, GRN and IL8. We, therefore, evaluated the expression levels of miR-223, involved in myeloid differentiation: these cases had significantly higher levels of miR-223 than had the other cases of T-cell acute lymphoblastic leukemia, with values comparable to those observed in acute myeloid leukemia. Finally, these patients appear to have an unfavorable clinical course. Conclusions Using gene profiling we identified a subset of adult T-cell acute lymphoblastic leukemia, accounting for 10% of the cases analyzed, which displays myeloid features. These cases were not recognized by standard approaches, underlining the importance of gene profiling in identifying novel acute leukemia subsets. The recognition of this subgroup may have clinical, prognostic and therapeutic implications.

Partial deletions of long arm of chromosome 6: biologic and clinical implications in adult acute lymphoblastic leukemia

Within 285 adult acute lymphoblastic leukemias (ALL) included in the multicenter GIMEMA 0496 trial and prospectively studied by conventional cytogenetics, 18 cases (6%) with long arm deletion of chromosome 6 (6q) were identified. These cases were divided into: (i) del(6q) only (n = 6); (ii) del(6q) plus other numerical and/or structural abnormalities (n = 8); (iii) del(6q) and other ‘specific’ translocations (n = 4). The biologic and clinical features of the patients carrying this anomaly, as well as their outcome, were compared with those of 267 patients without del(6q). A T cell phenotype was more frequently associated with del(6q) cases in general (P = 0.001) and particularly with cases presenting del(6q) as the isolated abnormality (P = 0.0027). No significant difference with respect to multidrug resistance (MDR)/P glycoprotein expression was observed between the two groups of patients (21% vs 28% of MDR-positive cases, respectively). A BCR-ABL fusion transcript was less frequently detected in cases with del(6q) (11%) compared with those without the anomaly (29%). p15 and p16 deletions were identified by Southern blot analysis in 21% of cases with del(6q) and in 26% of cases without del(6q). In this latter group, a T cell phenotype was less frequently associated with p15 and/or p16 deletion than in the group carrying del(6q) (36% vs 100% of cases, P = 0.011). Overall, patients with ALL and del(6q) had a high complete remission (CR) rate (83%); however, they had a lower 18 month event-free survival (31% vs 41%) and a higher relapse rate (70% vs 37%, P = 0.02) compared with patients without del(6q). To date, this is the largest series of adult ALL cases reported with del(6q) homogeneously treated, which have also been prospectively studied for MDR expression and for the detection of known fusion genes. This anomaly, as an isolated change, identifies a subset of cases with hyperleukocytosis (median WBC count 52 × 109/l) and a strict correlation with a T cell phenotype. Overall, del(6q) seems to be associated with an unfavorable clinical outcome, although this finding will need to be confirmed by extended FISH analysis.

Clinico-biological features of 5202 patients with acute lymphoblastic leukemia enrolled in the Italian AIEOP and GIMEMA protocols and stratified in age cohorts

The outcome of children and adults with acute lymphoblastic leukemia is markedly different. Since there is limited information on the distribution of clinico-biological variables in different age cohorts, we analyzed 5202 patients with acute lymphoblastic leukemia enrolled in the Italian multicenter AIEOP and GIMEMA protocols and stratified them in nine age cohorts. The highest prevalence of acute lymphoblastic leukemia was observed in children, although a second peak was recorded from the 4th decade onwards. Interestingly, the lowest incidence was found in females between 14–40 years. Immunophenotypic characterization showed a B-lineage in 85.8% of patients: a pro-B stage, associated with MLL/AF4 positivity, was more frequent in patients between 10–50 years. T-lineage leukemia (14.2%) was rare among small children and increased in patients aged 10–40 years. The prevalence of the BCR/ABL1 rearrangement increased progressively with age starting from the cohort of patients 10–14 years old and was present in 52.7% of cases in the 6th decade. Similarly, the MLL/AF4 rearrangement constantly increased up to the 5th decade, while the ETV6/RUNX1 rearrangement disappeared from the age of 30 onwards. This study shows that acute lymphoblastic leukemia in adolescents and young adults is characterized by a male prevalence, higher percentage of T-lineage cases, an increase of poor prognostic molecular markers with aging compared to cases in children, and conclusively quantified the progressive increase of BCR/ABL+ cases with age, which are potentially manageable by targeted therapies.

Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults

Background Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias. Design and Methods We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias. Results The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia. Conclusions Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

Umbilical cord blood transplant from unrelated HLA-mismatched donors in children with high risk leukemia

In the last 3 years, 14 children with high-risk leukemia (11 ALL, 2 AML and 1 CML) underwent cord blood transplantation from unrelated HLA-mismatched donors at a median of 99 days from the start of search. Eight patients were transplanted in second CR, one in accelerated phase, three at relapse and two patients in first CR. Conditioning regimen (fractionated TBI, etoposide, CY and anti-lymphocyte serum) and prophylaxis of GVHD (CsA and 6-methylprednisolone) were identical for all patients. Neutrophils > 0.5 × 109/l were reached at a median of 33 days from transplant, but in four cases we observed an autologous hematopoietic reconstitution (three spontaneous, one after autologous BM rescue). Acute and chronic GVHD were observed in 10/14 and 3/8 evaluable cases, respectively. Three patients died of transplant-related toxicity and three patients relapsed. The probabilities of event-free, disease-free and overall survival were 50, 53 and 64%, respectively. Cord blood transplant from HLA-mismatched unrelated donor is a valid option for the treatment of children with high-risk leukemia. With our eligibility criteria, conditioning regimen and prophylaxis of graft-versus-host disease, the main obstacles to successful transplant were represented by graft failure and fatal acute GVHD.

An accurate and rapid flow cytometric diagnosis of BCR-ABL positive acute lymphoblastic leukemia

This report describes an accurate and rapid flow cytometric diagnosis of BCR/ABL positive acute lymphoblastic leukemia. See related perspective article on page 1639. Tyrosine kinase inhibitors have profoundly modified the treatment and prognosis of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. A rapid and accurate detection of the BCR-ABL fusion protein is paramount today for an optimal management of Ph+ acute lymphoblastic leukemia. We have utilized a recently described and commercialized immunoassay that identifies qualitatively the presence of the BCR-ABL protein in leukemic cell lysates. The BCR-ABL fusion protein is captured and detected by a cytometric bead assay and analyzed by flow cytometry. The assay was applied to 101 primary patient samples (94 acute leukemias and 7 chronic myeloid leukemia blast crisis) and the results of the immunoassay were concordant with those obtained by conventional molecular techniques. The method proved reliable, reproducible, of simple execution and it was successfully completed within four hours. This flow cytometric immunoassay has important implications for perfecting the management of Ph+ acute lymphoblastic leukemia patients worldwide.