Carmen Baldazzi

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.

Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response, a study of the GIMEMA CML WP

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Chromosome abnormalities additional to the Philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications

Additional chromosome abnormalities (ACAs) occur in less than 10% of cases at diagnosis of Philadelphia chromosome (Ph)-positive chronic myelogenous leukemia (CML). In some cases, on the basis of the persistence of the ACAs in Ph-negative cells after response to imatinib, a secondary origin of the Ph chromosome has been demonstrated. In this study, the possible prognostic value of this phenomenon was evaluated. Thirty-six Ph-positive CML patients were included in the study. In six patients, ACAs persisted after the disappearance of the Ph. A complete cytogenetic response (CCR) was obtained in five of these six patients, and five of six also had a high Sokal score. In all the other cases, ACAs disappeared together (in cases of response to therapy with imatinib) or persisted with the Ph (in cases of no response to imatinib). In the former cases, the primary origin of the Ph was demonstrated. CCR was obtained in 22 cases (17 with low to intermediate Sokal scores), while no response was observed in 8 patients (5 with a high Sokal score). Sokal score seems to maintain its prognostic value for patients in whom the Ph occurs as a primary event, but not in those in whom it occurs as a secondary one.

Emergence of clonal chromosomal abnormalities in Philadelphia negative hematopoiesis in chronic myeloid leukemia patients treated with nilotinib after failure of imatinib therapy

Clonal cytogenetic abnormalities (CAs) in Philadelphia negaive (Ph−) metaphases have been widely observed during imatinib reatment in patients with chronic myeloid leukemia (CML) [1,2]. ecently, the appearance of such abnormalities in some CML atients, treated with dasatinib after imatinib failure, has been escribed [3,4]. To the best of our knowledge, only one patient has een reported to develop a cytogenetic abnormality in Ph− clone uring nilotinib treatment, following imatinib [5]. The incidence nd consequences of CAs in Ph− cells during treatment with second eneration tyrosine kinase inhibitors (TKIs) should be extensively nvestigated; this issue, deserves a particular attention, since these ew drugs put a far higher pressure on the leukemic and the residal normal hematopoiesis than imatinib. At our institute we analyzed 30 late chronic phase (CP) CML atients treated with nilotinib after imatinib failure by conventional ytogenetic (CC) and fluorescence in situ hybridization (FISH). Cytoenetic analysis of bone marrow (BM) was performed at diagnosis, uring imatinib treatment, before starting nilotinib and every 3–6 onths during the first year of nilotinib therapy; thereafter every 12 onths or in case of disease progression. FISH was performed using NA commercial probes. At diagnosis LSI Dual Color Dual Fusion CR-ABL DNA probe (Vysis, Downers Grove, IL, USA) was used to haracterize BCR-ABL rearrangement. The subsequent analysis was erformed using appropriate probes, depending on the results of C. The patients were 18 females and 12 males, median age was 1 years (range 18–77). The median time of imatinib therapy was 4 months (range 7–71). Fourteen patients received only imainib as previous therapy, whereas 16 patients were treated with dditional therapies, such as: hydroxyurea, interferon, cytosinerabinoside, busulphan. During nilotinib therapy 18 (60%) patients chieved a complete cytogenetic response (CCyR), within a median ime of 4.7 months (range 1–10). Patients were followed for a edian time of 18 months (range 4–42) after commencing nilotinib herapy (Table 1). Three out of 30 patients (10%) developed CAs in Ph− cells, amely: trisomy 8, del(20)(q11q13) and t(3;5)(p12;p13). There ere 2 males and 1 female, aged between 31 and 58 years. el(20q)(q11q13) (no.1) and trisomy 8 (no.2) persisted for 12 nd 18 months, respectively, and were still present at the last ytogenetic study (21 and 24 months, respectively), whereas (3;5)(p12;p13) (no.3) was transient: it appeared after 6 months f therapy and disappeared within 2 months. Patients’ characeristics are summarized in Table 2. Retrospective FISH analysis erformed on stored BM specimens of patients no.1 and no.2 ailed to detect the above reported aberrations before niloinib therapy. After a median follow-up of 19 months (range

Influence of additional cytogenetic abnormalities on the response and survival in late chronic phase chronic myeloid leukemia patients treated with imatinib: long-term results

Department of Hematology/Oncology ‘‘L. and A. Seràgnoli’’ S. Orsola Malpighi Hospital, University of Bologna, Bologna, Italy, Department of Cellular Biotechnology and Hematology, University ‘‘La Sapienza’’, Rome, Italy, Hematology Section, University of Bari, Bari, Italy, CEINGE Biotecnologie Avanzate and Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy, and Department of Clinical and Biological Science, University of Turin at Orbassano, Turin, Italy

Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients

FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

Acute promyelocytic leukemia with amplification of PML-RARα rearrangement: Clinical implications

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Abstract 906: Gas1 and Kif27 genes are strongly up-regulated biomarkers of Hedgehog inhibition (PF-04449913) on leukemia stem cells in Phase I Acute Myeloid Leukemia and Chronic Myeloid Leukemia treated patients

Hedgehog (Hh) pathway activation contributes to leukemia development and growth, and that targeted pathway inhibition is likely to offer an efficient therapeutic opportunity. PF-04449913, a Hh pathway inhibitor, is a new selective and potent inhibitor of leukemia self-renewal and is currently being evaluated in phase I clinical trials. In order to identify new potential clinical biomarkers for the PF-04449913, we studied CD34+ leukemia stem cell population (LSC) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies. This experimental clinical trial enrolled Myelofibrosis (MF), MDS, blastic phases CML, chronic myelomonocytic leukemia (CMML) and AML patients (pts). We were able to collect and separate highly purified (98%) bone marrow CD34+ cells from 5 AML, 1 MF and 2 CML pts by immunomagnetic separation, and analysed them for gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Clustering of their expression profiles showed that mostly genes differentially expressed are mainly related to Hh signaling, this providing further evidences that PF-04449913 really therapeutically targets the Hh pathway. Regarding genes involved in Hh signaling pathway, Gas1 and Kif27 were strongly upregulated (fold change 1.0947 and 1.12757 respectively; p-value 0.01 and 0.02 respectively) in CD34+ LSC after 28 days exposure to PF-04449913 as compared to baseline, suggesting these two genes have potential as new biomarkers of activity. GAS-1 is a Sonic Hedgehog (Shh)-binding protein; it acts to sequester Shh and inhibit the Shh signalling pathway. Kif27 mainly acts as a negative regulator in the Hh signaling pathway, and inhibits the transcriptional activator activity of Gli1 by inhibiting its nuclear translocation. Other genes were differentially expressed after ‘ex- vivo’ treatment with PF-04449913 as compared to baseline: we observed a down regulation of Bcl2 (fold change -1.03004), ABCA2 (fold change -1.08966), LEF1 (fold change -1.28457), Gli1 (fold change -1.0775), Smo (fold change -1.07702), and an upregulation of Gli2 (fold change 1.08191). Conclusions: This data demonstrates that PF-04449913 specifically targets the Hh Pathway in CD34+ cells, suggesting that Hh inhibition may impair leukemia stem cell maintenance. In addition, we identify several new potential biomarkers (e.g. Gas1 and KIF27). Taken together, these data may be useful for pts selection strategies and subsequent eradication of the LSC. Acknowledgments: Work supported by Pfizer, European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 906. doi:1538-7445.AM2012-906

Abstract 746: PKC412 (Midostaurin) is safe and highly effective in systemic mastocytosis patients: Follow up of a single-center Italian compassionate use

Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs. The recent WHO classification (2008) includes an indolent form (ISM), an aggressive form (ASM) and a leukemic subvariant (MCL). The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 22 patients (male/female=11/11) affected by SM have been referred to our Institution. Among these, 12 (55%) presented with systemic symptoms associated with signs of organ involvement (skeletal lesions, ascites, liver function impairment or bon marrow disfunction), thus identifying an ASM. Therefore, since a first line therapy (IFNalfa, Imatinib and 2CdA in 56%, 11% and 33% of the patients, respectively) and supportive care with histamine receptor antagonists weren’t followed by a significant benefit, a personalized use of PKC412 was asked and obtained for 9 out of 12 ASM patients. Thus, from March 2011 9 (M/F =3/9) patients with ASM have been treated with PKC412, administered orally, at the dosage of 100 mg twice daily, without rest periods. The median age was 60 years (range 39-75); the median time from diagnosis was 6 months (range 2-53). Median serum tryptase level was 100 mcg/L(range 19.3-1160). C-kit mutation D816V was present in 8 out of 9 patients. Cytogenetic analysis was normal in all the patients. Results. Seven out of nine patients were evaluable for response. The median duration of therapy was 517 days (range 327-970+). According to European Criteria, a Major response was observed in one patient, and a partial response in 6 patients. Overall, the drug was well tolerated, and no serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. Conclusions. In a small cohort of ASM patient, the prolonged therapy with PKC412 is safe and effective, mainly on symptoms improvement and haematological profile. Nevertheless, the persistence of the D816V c-kit mutation, despite significant responses, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. UDS approaches are needed in order to clarify this issue. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN 2010-2011, University of Bologna, FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Simona Soverini, Caterina De Benedittis, Maria Chiara Abbenante, Chiara Sartor, Ilaria Iacobucci, Carmen Baldazzi, Emanuela Ottaviani, Anna Ferrari, Viviana Guadagnuolo, Alberto Conficoni, Strefania Paolini, Sarah Parisi, Federica Frabetti, Silvia Piccari, Sandro Grilli, Elisa Lani, Giovanni Martinelli. PKC412 (Midostaurin) is safe and highly effective in systemic mastocytosis patients: Follow up of a single-center Italian compassionate use. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2014-746

Mesenchymal stromal cells from myelodysplastic and acute myeloid leukemia patients display in vitro reduced proliferative potential and similar capacity to support leukemia cell survival

BackgroundMesenchymal stromal cells (MSCs) are an essential element of the bone marrow (BM) microenvironment, playing a crucial function in regulating hematopoietic stem cell proliferation and differentiation. Recent findings have outlined a putative role for MSCs in hematological malignancy development. So far, conflicting results have been collected concerning MSC abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). In particular, a considerable amount of evidence has been accumulated strongly supporting a permissive role of MSCs in malignancy evolution to MDS, while a potentially causative or promoting function performed by MSCs in AML has not yet been fully clarified. Here, we compared MSCs isolated from healthy, MDS, and AML subjects to investigate MSC alterations and to emphasize putative common and/or diverse features.MethodsWe isolated and expanded MSCs from AML patients (AML-MSCs) and MDS patients (MDS-MSCs), and we analyzed and compared their phenotypic and functional properties with respect to each other and versus healthy donor-derived MSCs (HD-MSCs).ResultsWe found that stable MSC cultures could be easily established from HD and MDS mononuclear BM-derived cells, while a substantial fraction (25%) of AML patients failed to yield MSCs. Nevertheless, isolated MDS-MSCs and AML-MSCs, as well as HD-MSCs, contained the basic features of MSCs. Indeed, they displayed similar surface marker expression and efficient capacity to differentiate versus osteogenic and adipogenic lineage in vitro. We also proved that MDS-MSCs and AML-MSCs, analyzed by fluorescence in-situ hybridization, did not harbor leukemic cell cytogenetic abnormalities. Moreover, MDS-MSCs and AML-MSCs were similar in terms of ability to sustain AML cell viability and immune-regulatory capacity. However, we were also able to detect some differences between AML-MSCs and MDS-MSCs. Indeed, we found that the frequency of rescued MSCs was lower in the AML group than in the HD and MDS groups, suggesting that a reduced number of MSC precursors could inhabit AML BM. Instead, MDS-MSCs showed the lowest proliferative capacity, reflecting some intrinsic and particular defect.ConclusionsOverall, our results elucidated that MDS-MSCs and AML-MSCs did not show macroscopic and/or tumor-related defects, but both displayed functional features potentially contributing to favor a leukemia-protective milieu.