Antonia Rotolo

Emerging drugs for chronic myeloid leukemia

Importance of the field: The deregulated tyrosine kinase activity of BCR-ABL has been demonstrated to be necessary and sufficient to maintain leukemia phenotype of chronic myeloid leukemia (CML) which, therefore, represents a unique model for the development of molecular targeted therapy and the first disease in which the tyrosine kinase inhibitors (TKIs) completely changed the therapeutical approach. The impressive results of TKIs in this model have been overshadowed by the development of clinical resistance. Areas covered in this review: This review focuses on clinical results with imatinib therapy and second generation TKIs. Furthermore, a summary of the guidelines for the management of TKI resistant patients is provided together with a description of the new drugs in clinical or preclinical phases which are developing to overcome resistance. What the reader will gain: Future perspective for the ‘cure’ of CML patients and new drugs designed for this purpose are suggested. Take home message: CML therapy has dramatically changed in the last few years due to the introduction of targeted therapy. Studies on new drugs targeting different pathways other than BCR-ABL are ongoing to improve the clinical results.

Detection of humoral immune responses against WT1 antigen in patients affected by different hematological malignancies.

(MM) patients suggests an evident clinical benefit, although the WT1 gene is not overexpressed in their cells. Furthermore, it was clearly demonstrated that MM cells are highly sensitive to WT1-specific cytotoxic T lymphocytes [15] . In vitro and in vivo evidence in many hematological disorders suggests the possibility that the humoral immune responses towards WT1 protein could be elicited, too. To verify this hypothesis, we have analyzed the presence of WT1 antibodies in a large cohort of untreated patients affected by different types of hematological malignancies. Furthermore, in order to establish whether WT1 expression could predict the immune response and to calculate the level of correlation between WT1 mRNA and antibodies, we tested patients with high, low or absent levels of WT1 transcript. Finally, we tested a group of healthy subjects since few data are available regarding their WT1 antibody level [14] . To address this point, serum samples were collected at diagnosis from 139 patients affected by the following hematological disorders: 25 AML, 40 MDS [12 refractory The Wilms tumor gene (WT1) is highly expressed in many types of hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML) and Ph-negative myeloproliferative disorders such as idiopathic myelofibrosis (IMF), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndromes or chronic neutrophilic leukemia [1–5] . WT1 overexpression prompted many researchers to develop vaccine strategies using WT1 as immunological target [6–10] . The clinical results, although still preliminary, are encouraging in terms of feasibility, safety and efficacy although this latter point requires strategy improvements being at a very early phase of development [6–13] . One important step could the adequate selection of patients who are candidates for vaccine protocols. Few years ago, the presence of humoral immune responses against the WT1 product was reported in a cohort of patients affected by AML, MDS and CML [14] . The presence of WT1 antibodies in the serum of leukemia patients is relevant in view of the development of vaccination approaches since it demonstrates that WT1 protein can be recognized by the immune system. Interestingly, WT1 peptide vaccination in multiple myeloma Received: January 15, 2008 Accepted after revision: June 25, 2008 Published online: September 30, 2008

Herpetic leg paralysis and abdominal ileus in a patient with idiopathic myelofibrosis

A 78-year-old man was referred to our department because of the sudden onset of motor paralysis of the legs (Fig. 1). The patient reported a sudden fall to the ground the day before admission 4 years prior, he had been diagnosed with idiopathic myelofibrosis (PMF) in the hypercellular phase. He was currently under hydroxiurea based cytoreductive therapy. The past medical history included atrial fibrillation still in therapy with oral anticoagulants and digitalis. Over the previous month, the patient reported the onset of lower right limb pain with weakness. The physical examination was positive for fever and a scattered vesicular eruption of the lower half of the right limb involving the knee and the thigh. Blood tests revealed severe anemia and leucopenia, immediately treated with a prophylactic wide-spectrum antibiotic, granulocyte colony-stimulating factor (G-CSF) and erythrocyte transfusions. Blood cultures were negative. X-ray studies of the chest and of the right femur and pelvis were normal. Magnetic resonance was not performed because the patient was wearing a pacemaker. Brain computed tomography (CT scan) was normal, without any evidence of active encephalytic lesions. Since the vesicular eruption was suggestive for herpes zoster infection, a lumbar puncture was performed showing the presence of lympho-monocytosis, hyperproteinorrachia and hypoglycorrachia. Spinal fluid analysis for the detection of Varicella zoster virus (VZV) by real time polymerase chain reaction was highly positive (49,309 copies/ml). Therapy with high-dose intravenous acyclovir (10 mg/kg 9 3/day) was immediately started. During treatment he developed a major genital oedema and abdominal distention. The abdominal X-ray (Fig. 2) and CT scan showed multiple fluid levels suggestive of ileus. After 1 day, the patient presented urinary retention with bladder distention, dyspnea with a persistent irritative cough prompting a repeat chest X-ray study, which showed ground glass features (Fig. 3). Blood gas analysis revealed: pH: 7.35; PaCO2: 40 mmHg; PaO2: 54 mmHg; HCO3 : 21 mmol/L. This clinical picture can be associated with a widespread VZV infection involving the central nervous system (CNS). Indeed, it is documented that VZV infection at the CNS level can produce transient ischemic attack (TIA) or a stroke in adults [1], suggesting that VZV encephalitic vasculopathy could represent the cause for the right limb paralysis. The bowel and bladder paralysis are probably related to persistent VZV infection in the autonomic ganglia. It is reported that the intestinal pseudo-obstruction can be directly caused by VZV infection in the immunodeficient host (Ogilvie’s syndrome) [2]. After 1 week of treatment with acyclovir, a slow and gradual resolution of all the symptoms was observed. This clinical response to the treatment and the presence of a demonstrated VZV infection correlate with observed complications of a systemic herpes zoster infection. Nonetheless, after two further weeks, a progressive worsening of the clinical conditions, complicated by pulmonary bacterial infection, resulted in the patient’ death. Herpes zoster is a common vesicular eruption due to the reactivation of latent VZV in the dorsal sensory ganglia. Herpes zoster occurs at some point in the lifetime in 10–20% of the population [3]. The most common clinical presentation is represented by a painful cutaneous rash, Paolo Nicoli and Marco Bosa contributed equally to this paper.

Abstract 251: Disabled gene is involved in CML progression and its expression level at diagnosis can predict major molecular response (MMR) to imatinib therapy

Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. Using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we identified Dab1 and Dab2, the homologs of Dm Disabled (Dab), as genes involved in CML progression. In Dm the Dab loss of function induced a worsening of the hBcr-Abl eye phenotype and an even stronger phenotype was obtained using Dab RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Dab is an adaptor protein acting downstream of many receptor tyrosine kinases (RTK). One of the human homolog of Dab, Dab1 is a large common fragile site gene involved in neural migration, and the other homolog Dab2 encodes an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many cancers suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM) distributed as follows: 55 patients at diagnosis (19 enrolled in TOPS study), 9 chronic phase (CP), 7 accelerated phase (AP) and 11 blast crisis (BC). 21 healthy donors (10 PB and 11 BM) were analyzed as control. In 18 patients, genes expression was analyzed during remission as well. Protein expression was evaluated by Western Blot (WB) and Immunofluorescence (IF). In addition, K562 cells were transfected with Dab plasmids to evaluate the effects on cell proliferation. We found that in CML patients Dab1/2 expression was significantly decreased both in BM and PB (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 251.

Abstract 246: Genome-wide screening for dominant modifiers in Drosophila identified new genes involved in BCR-ABL signaling and chronic myeloid leukemia (CML) progression

Despite the role of Bcr-Abl in the pathogenesis of CML is well established, the mechanisms responsible for CML progression remain unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed the genetic screening using our set-up model of human p210 Bcr-Abl (hBcr-Abl) transgenic Drosophila melanogaster (Dm) in which the tissue specific expression of hBcr-Abl is able to induce a severe eye phenotype or the formation of melanotic tumors, when expressed into the fly lymph gland, the Dm hematopoietic system. The whole fly genome containing 14.000 genes was analyzed using 278 fly stocks carrying well characterized chromosome deletions. The resulting progeny was screened using eye and lymph gland as first and second read-out system respectively. Data obtained from both screens were analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified genes were validated analyzing either BM or PB samples from CML patients and healthy donors. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screen crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. 42% of the 4000 Dm genes mapping in these regions displayed a known human counterpart. Gene Ontology profiles of these genes included oncogenes, tumor suppressors and genes involved in the regulation of transcription, signal transduction, proliferation, differentiation, apoptosis and splicing. Moreover, a computational comparison of our results in fly with gene expression signatures of CML from Microarray data, showed only a partial overlap. Interestingly, the 72% of identified genes was not known to be involved in human leukaemia. However, further confirmation of our findings comes from the validation in human samples in which 1250 genes were found to be associated with CML; among these genes, we found not only an alteration of their expression profiles in CML patients compared to the healthy donors, but also protein alterations such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study points to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 246.

New drugs in the treatment of chronic myeloid leukaemia

The introduction of the BCR-ABL kinase inhibitor, imatinib mesylate (Gleevec®, Novartis) led to significant changes in the treatment of chronic myeloid leukaemia (CML) patients. However, despite the impressive percentage of responding patients, some CML cases, particularly those in advanced phases of the disease, show primary resistance or relapse after the initial response. The second-generation BCR-ABL inhibitors nilotinib (Tasigna®, Novartis) and dasatinib (Sprycel®, Bristol-Myers Squibb) have shown significant activity in clinical trials in patients who failed imatinib therapy, but these agents are still incapable of inhibiting the T315I mutant of Bcr-Abl and present partial activity in advanced phases of CML. The acquired biological notions of the mechanisms of tyrosine kinase inhibitor (TKI) resistance has led to the development of new compounds, some of which have shown encouraging preliminary results in clinical trials, even against T315I mutants. In this paper we discuss the new emerging therapies which may overcome TKI resistance in CML patients.