Maria Chiara Abbenante

CDKN2A/B Alterations Impair Prognosis in Adult BCR-ABL1–Positive Acute Lymphoblastic Leukemia Patients

Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases. Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1–positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. ©2011 AACR.

Complete paraplegia after nelarabine treatment in a T-cell acute lymphoblastic leukemia adult patient.

A 30–year-old man was diagnosed with ALL T-lineage immunophenotype, (CD71, CD131, CD451, MPO-), normal karyotype, with no CNS involvement (negative lumbar puncture) and treated with a cycle of induction chemotherapy, including Vincristine, L-Asparaginase, and Adriamycin, with complete morphological remission. Two consolidation cycles were then administered, followed by maintenance chemotherapy with oral 6-Mecaptopurine (6-MP) and Methotrexate (MTX). As a matched bone marrow donor was not detected, the patient could not receive a stem cells transplantation. Hence, as expected, three years after diagnosis, because of the development of hyperleukocytosis (WBC 22,500/mm) a first relapse was documented, with the presence of translocation (5;14) in 30 out of 30 metaphases. The patient underwent four cycles of savage chemotherapy, obtaining a second complete morphologic and cytogenetic remission after which he was treated with steroids, MTX, and 6-MP, as maintenance. Six months later, a second relapse occurred, and he received a cycle of chemotherapy with Nelarabine at the adult standard dosage (1,500 mg/sqm/daily on days 1, 3, and 5), which was well-tolerated. The iatrogenic bone marrow aplasia was complicated by two febrile episodes, fully resolved with antibacterial therapy, and by liver toxicity (Grade III according to WHO classification) characterized by increased cytonecrosis indexes. As a complete remission was documented, 28 days later the patient received a second cycle of Nelarabine, at the same dosage. On the third day of therapy, the patient reported the onset of severe paresthesias in his legs, bilaterally, rapidly extended to the anterior abdominal wall and to the gluteus skin. Moreover, he revealed a significant defect in maintaining equilibrium and a reduced ability in walking. A neurologic detailed examination showed a strong-force defect, especially involving legs, and a severe difficulty in standing. Lasègue sign was negative, whereas Babinski sign was detected, bilaterally. Neither force nor sensitivity defects were found at his upper arms. Alterations of Image 1. Sagittal T2 image of the thoracic spine demonstrates abnormal hypointense marrow signal in the vertebral bodies consistent with leukemic infiltration. In addition, there is abnormal hyperintense T2 signal in the mid-thoracic spinal cord at T5 consistent with transverse myelitis (arrow). urinary and bowel functions were also reported. Based on the clinical suspicion of a massive spinal cord pressure, a lumbo-sacral MRI was immediately performed, which showed, at the level of the fifth dorsal vertebra, an alteration of the intramarrow signal, with longitudinal extension of 2 cm, and anteroposterior and lateral diameter of 0.6 and 0.8 cm, respectively (Image 1). This radiological image was consistent with transverse acute myelitis; a lumbar puncture was performed, which was negative for both CNS leukemic and infective involvement, and for ischemic damage. Therefore, the patient received steroid intravenous therapy, with only a mild and transitory improvement of the paresthesias, and no change on equilibrium and autonomic defects. A MRI with an angiographic detailed study, repeated seven days later, showed no changes. These symptoms did not further improve despite intensive rehabilitation physiotherapy. The patient maintained complete morphologic remission for 8 months, without therapy. Having ruled out ischemic and hemorrhagic etiology, and a leukemic infiltration, the worsening of sensory and motor defects and the rapid loss of autonomic functions were most likely related to cumulative drug toxicity, mainly ascribable to the administration of Nelarabine. Nelarabine is a new purine analogue, approved by FDA for the treatment of patients with T-ALL and T-cell lymphoblastic lymphoma (T-LBL) whose disease has not responded to or has relapsed following at least two chemotherapy regimens. Neurologic toxicity is dose limiting for both pediatric and adult patients [1]. Major neurologic adverse events in adult population, usually include Grade I–II somnolence, dizziness, hypoesthesia, headache, paresthesia, and depressed level of consciousness. So far, rare cases of Grade III peripheral neurological disorders or ataxia have been described. Despite the high efficacy of Nelarabine [2], neurological toxicity should always be considered and carefully monitored during treatment. To the best of our knowledge, we have reported here the first case of a T-ALL patient with a lasting hematological response induced by Nelarabine, which was associated with severe and irreversible neurological toxicity, consistent with a complete paraplegia.

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positive acute lymphoblastic leukemia in complete response

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph−) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph− ALL.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Rationale for a pediatric-inspired approach in the adolescent and young adult population with acute lymphoblastic leukemia, with a focus on asparaginase treatment

In the last two decades great improvements have been made in the treatment of childhood acute lymphoblastic leukemia, with 5-year overall survival rates currently approaching almost 90%. In comparison, results reported in adolescents and young adults (AYAs) are relatively poor. In adults, results have improved, but are still lagging behind those obtained in children. Possible reasons for this different pattern of results include an increased incidence of unfavorable and a decreased incidence of favorable cytogenetic abnormalities in AYAs compared with children. Furthermore, in AYAs less intensive treatments (especially lower cumulative doses of drugs such as asparaginase, corticosteroids and methotrexate) and longer gaps between courses of chemotherapy are planned compared to those in children. However, although favorable results obtained in AYAs receiving pediatric protocols have been consistently reported in several international collaborative trials, physicians must also be aware of the specific toxicity pattern associated with increased success in AYAs, since an excess of toxicity may compromise overall treatment schedule intensity. Cooperative efforts between pediatric and adult hematologists in designing specific protocols for AYAs are warranted.

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.

Recurrent Gastrointestinal Hemorrhage in Treatment with Dasatinib in a Patient Showing SMAD4 Mutation with Acute Lymphoblastic Leukemia Philadelphia Positive and Juvenile Polyposis Hereditary Hemorrhagic Telangiectasia Syndrome

We report a case of a patient affected by juvenile polyposis and hereditary hemorrhagic telangiectasia linked to a SMAD4 mutation who developed acute lymphoblastic leukemia positive for the Philadelphia chromosome translocation and with a complex karyotype. During the treatment with the tyrosine kinase inhibitor dasatinib the patient presented recurrent severe gastrointestinal hemorrhages linked to the genetic background and aggravated by thrombocytopenia.

A Case Report of Acute Myeloid Leukemia and Neurofibromatosis 1

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the NF1 gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult.

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