Emanuela Ottaviani

Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP)

The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukemia (ALL) with the worst clinical prognosis. To identify oncogenic lesions that combine with BCR-ABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0), fluorescence in situ hybridization, and genomic polymerase chain reaction to study 106 cases of adult BCR-ABL1-positive ALL. The most frequent somatic copy number alteration was a focal deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 (75%) of 106 patients. Different patterns of deletions occurred, but the most frequent were those characterized by a loss of exons 4 through 7 (Delta4-7) and by removal of exons 2 through 7 (Delta2-7). A variable number of nucleotides (patient specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the Delta4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localization and oncogenic activity, whereas the Delta2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion also was identified in the progression of chronic myeloid leukemia to lymphoid blast crisis (66%) but never in myeloid blast crisis or chronic-phase chronic myeloid leukemia or in patients with acute myeloid leukemia. Known DNA sequences and structural features were mapped along the breakpoint cluster regions, including heptamer recombination signal sequences recognized by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination.

Molecular Remission After Allogeneic or Autologous Transplantation of Hematopoietic Stem Cells for Multiple Myeloma

PURPOSE To assess the clinical relevance of minimal residual disease (MRD) in patients with multiple myeloma (MM), 50 patients were monitored while they were in complete clinical remission (CCR) after autologous or allogeneic stem-cell transplantation. PATIENTS AND METHODS Stringent molecular monitoring using clonal markers based on rearranged immunoglobulin heavy-chain genes was performed in 44 of 50 MM patients in CCR. Molecular clinical remission (MCR) was defined as more than one consecutive negative polymerase chain reaction (PCR) test result. RESULTS Twelve (27%) of 44 molecularly monitored patients achieved MCR; four of the 12 became PCR-positive, and one of these four relapsed. In comparison with patients who did not achieve MCR, patients who achieved MCR had a significantly lower relapse rate (41% v 16%; P <.05) and longer relapse-free survival (35 v 110 months; P <.005). Fourteen of 26 patients in CCR who had received allografts were evaluated on a molecular basis: seven (50%) of the 14 achieved MCR and did not relapse; one of the seven remaining patients relapsed. Thirty of 47 patients in CCR who received autografts were evaluated on a molecular basis: five (16%) of the 30 achieved MCR; two of these five became PCR-negative, and one of these two relapsed. Ten of the 25 remaining patients later relapsed. For these nonrandomized groups, the higher MCR rate after allograft procedures was statistically significant (P <.01; Fishers exact test). CONCLUSION MCR can be obtained in a relatively high proportion of MM patients who have achieved CCR after undergoing allograft procedures and in a smaller fraction of patients after undergoing autograft procedures. In approximately one fourth of MM patients who achieve CCR after transplantation, it may be possible to keep the disease burden constantly below the PCR threshold. Because MCR was associated with prolonged relapse-free survival, these patients could have a relatively favorable clinical outcome.

The efficacy of imatinib mesylate in patients with FIP1L1-PDGFRα-positive hypereosinophilic syndrome. Results of a multicenter prospective study

Background and Objectives The hypereosinophilic syndrome (HES) may be associated with the fusion of the platelet derived growth factor receptor α (PDGFRα) gene with the FIP1L1 gene in chromosome 4 coding for a constitutively activated PDGFRα tyrosine kinase. These cases with FIP1L1-PDGFRα rearrangement have been reported to be very sensitive to the tyrosine kinase inhibitor imatinib mesylate. Design and Methods A prospective multicenter study of idiopathic or primary HES was established in 2001 (Study Protocol Registration no. NCT 0027 6929). One hundred and ninety-six patients were screened, of whom 72 where identified as having idiopathic or primary HES and 63 were treated with imatinib 100 to 400 mg daily. Results Twenty-seven male patients carried the FIP1L1-PDGFRα rearrangement. All 27 achieved a complete hematologic remission (CHR) and became negative for the fusion transcripts according to reverse transcriptase polymerase chain reaction (RT-PCR) analysis. With a median follow-up of 25 months (15–60 months) all 27 patients remain in CHR and RT-PCR negative, and continue treatment at a dose of 100 to 400 mg daily. In three patients imatinib treatment was discontinued for few months, the fusion transcript became rapidly detectable, and then again undetectable upon treatment reassumption. Thirty-six patients did not carry the rearrangement; of these, five (14%) achieved a CHR, which was lost in all cases after 1 to 15 months. Interpretation and Conclusions All patients meeting the criteria for idiopathic or primary HES should be screened for the FIP1L1-PDGFRα rearrangement. For all patients with this rearrangement, chronic imatinib treatment at doses as low as 100 mg daily ensures complete and durable responses.

Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4–11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34+ cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K–Akt survival pathway.

Expression of spliced oncogenic Ikaros isoforms in Philadelphia-positive acute lymphoblastic leukemia patients treated with tyrosine kinase inhibitors: implications for a new mechanism of resistance

Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The expression of short isoforms lacking DNA-binding motifs alters the differentiation capacities of hematopoietic progenitors, arresting lineage commitment. We sought to determine whether molecular abnormalities involving the IKZF1 gene were associated with resistance to tyrosine kinase inhibitors (TKIs) in Ph+ acute lymphoblastic leukemia (ALL) patients. Using reverse-transcribed polymerase chain reaction, cloning, and nucleotide sequencing, only the non-DNA-binding Ik6 isoform was detected in 49% of Ph+ ALL patients. Ik6 was predominantly localized to the cytoplasm versus DNA-binding Ik1 or Ik2 isoforms, which showed nuclear localization. There was a strong correlation between nonfunctional Ikaros isoforms and BCR-ABL transcript level. Furthermore, patient-derived leukemia cells expressed oncogenic Ikaros isoforms before TKI treatment, but not during response to TKIs, and predominantly at the time of relapse. In vitro overexpression of Ik6 strongly increased DNA synthesis and inhibited apoptosis in TKI-sensitive cells. Genomic sequence and computational analyses of exon splice junction regions of IKZF1 in Ph+ ALL patients predicted several mutations that may alter alternative splicing. These results establish a previously unknown link between specific molecular defects that involve alternative splicing of the IKZF1 gene and the resistance to TKIs in Ph+ ALL patients.

Effects of homoharringtonine alone and in combination with alpha interferon and cytosine arabinoside on 'in vitro' growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoietic progenitors

Homoharringtonine (HHT) is a cephalotaxine alkaloid that showed clinical efficacy in the chronic phase of chronic myeloid leukemia (Ph1+CML). As a single agent, it resulted in effectively controlling leukocytosis and in producing sporadic karyotypic conversions; its clinical use in combination with interferon (IFN-α) for the treatment of CML could thus be considered. In this study we evaluated the growth inhibition and the induction of apoptosis determined by HHT alone and in combination with IFN-α and cytosine arabinoside (Ara-C) on normal and CML (both in chronic, CML-CP and in blastic phase; CML-BP) hematopoietic progenitors. HHT is able to determine a dose-dependent cell growth inhibition; evaluation of cytotoxic activity on semisolid cultures showed an activity significantly higher on CML-CP than on normal cells (P = 0.02 for HHT 50 ng/ml and P = 0.01 for HHT 200 ng/ml). HHT exerted a synergistic effect with IFN-α, Ara-C and IFN-α + Ara-C in inhibiting CML-CP colony growth; the same activity was demonstrated by the combination of HHT with Ara-C and by the triple combination, but not by HHT + IFN-α, on normal myeloid progenitors. The triple combination only was able to exert a synergistic effect in CML-BP. The induction of apoptosis resulted HHT dose-dependent in CML-CP and normals; at higher drug concentrations (100–200–1000 ng/ml), HHT induced a significant increase of apoptotic cells (for normals: P = 0.04, P = 0.02 and P = 0.04; for CML-CP: P = 0.01, P = 0.01 and P = 0.04, respectively); no significant changes were observed in CML-BP. In conclusion, the differences in cytotoxic effect and apoptosis induction observed, depending on the various phases of CML, add experimental evidence to the different clinical results between the chronic phase, where the clone is responsive to HHT, and the acute phase, where the drug is ineffective. The in vitro synergism of HHT with Ara-C and IFN-α in CML-CP suggests further evaluation in the clinical setting.

Nuclear translocation of protein kinase C-α and -ζ isoforms in HL-60 cells induced to differentiate along the granulocytic lineage by all-trans retinoic acid

We investigated whether members of the protein kinase C (PKC) family of enzymes were involved in the nuclear events underlying granulocytic differentiation induced by 10−6 M all‐trans retinoic acid (ATRA) in HL‐60 cells. PKC activity was analysed by using a serine substituted specific peptide which enabled the evaluation of the whole catalytic activity of both Ca++‐dependent and Ca++‐independent PKC isoforms. In parallel, the subcellular distribution of various PKC isoforms was evaluated by Western blot, immunoprecipitation and in situ immunocytochemistry analyses.

Long-term follow-up of 386 consecutive patients with essential thrombocythemia: Safety of cytoreductive therapy†

Cytotoxic agents like Hydroxyurea, Busulfan and Interferon‐alpha are to date the most commonly used therapeutic approaches in Essential Thrombocythemia (ET). However, few data on the efficacy and safety of these agents in the long‐term are currently available. We report a retrospective analysis of the long‐term outcome of 386 consecutive ET patients, followed at single Institution for a median follow‐up of 9.5 years (range, 3–28.5). Cytoreductive therapy was administered to 338 patients (88%), obtaining a response in 86% of cases. Forty‐five patients (12%) experienced a thrombosis. Among baseline characteristics, only history of vascular events prior to ET diagnosis predicted a higher incidence of thrombosis. Evolution in acute leukemia/myelofibrosis occurred in 6 (1,5%) and 20 (5%) patients, and was significantly higher in patients receiving sequential cytotoxic agents. Overall survival was 38% at 19 years and was poorer for patients older than 60 years, with higher leukocytes count (>15 × 109/L), hypertension and mellitus diabetes at ET diagnosis and for patients experiencing a thrombotic event during follow‐up. Cytoreductive therapy was effective in decreasing platelet number with negligible toxicity; however, thrombocytosis control did not reduce the incidence of thrombosis and, for patients who received sequential therapies, the probability of disease evolution was higher and survival was poorer. Am. J. Hematol. 2009.

A polymorphism in the chromosome 9p21 ANRIL locus is associated to Philadelphia positive acute lymphoblastic leukemia

Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and of MDM2 inhibitor p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed by genotyping 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions, in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph(+)) acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) samples, and 183 healthy controls. rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20-3.33; p=7.1×10(-3)). We hypothesized that this association reflects the capability of some ANRIL polymorphisms to contribute to its transcription changes responsible for alterations of CDKN2A/B expression profiles, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility.

Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells

Background The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. Design and Methods Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. Results We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4+CD25+ Foxp3+ T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4+CD25+ T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms’ tumor protein. Conclusions These data identify indoleamine 2,3-dioxygenase-mediated catabolism as a tolerogenic mechanism exerted by leukemic dendritic cells and have clinical implications for the use of these cells for active immunotherapy of leukemia.