PRESUBMISSION RESEARCH PLAN Foà Roberto [PI] Genetics-driven targeted management of lymphoid malignancies Keywords: Leukemias, Lymphomas, Diagnosis, Prognosis, Target therapy Background Lymphoid malignancies are among the most frequent neoplasms in Western countries, with acute lymphoid leukemia (ALL) being the commonest malignancy in children. At present, lymphoid neoplasms represent the third cancer in terms of frequency, but the WHO estimates that in about 20 years they will be the first. In a state-of-the-art hematologic setting, the laboratory and the clinic concur to provide a rapid and accurate diagnostic work-up, differential diagnosis among akin disorders, reliable prognostic stratification and minimal residual disease (MRD) monitoring, suggestion of targeted therapies, integration of the latter in the clinical management of patients and, ultimately, an improved quality of life and overall survival. Breakthroughs in the treatment of hematological neoplasms have stemmed from the identification of their underlying driving genetic lesion(s). The most illuminating examples are represented by acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML). In APL, understanding the driving genetic lesion has led to the clinical use of ATRA, that bypasses the differentiation block of the leukemic cells. Similarly, in CML the genetic-driven design of tyrosine kinase inhibitors (TKI) has changed the natural history, management and outcome of this disease. The use of TKI has recently been extended to Ph+ ALL. These examples highlight the importance of a genetics-driven molecular understanding of cancer and unequivocally document the feasibility in hematology of bench-bedside translation and its profound clinical impact. To make this progress possible a complex and highly skillful network is required that assembles diverse laboratories and clinical expertise, and state-of-the-art as well as new technologies, from diagnostic hematopathology and tissue banking to next-generation sequencing, molecular biology dissection of oncogenic pathways and clinical testing of new drugs. The program aims at broadening the genetics-driven targeted management of patients with lymphoid malignancies. The consortium is represented by internationally recognized experts in the molecular, diagnostic and clinical management of such malignancies. RF, SP and BF are also part of the steering committee for the WHO classification of hemopoietic neoplasms. In line with this, they work in (and were responsible of the set-up of) institutions that are referral centers for virtually all leukemias and lymphomas in Italy. Aims of the program The unifying and strongly mechanism-based concept of the current proposal is the discovery of the key genetic mechanisms underlying selected lymphoid malignancies and the exploitation of such mechanisms to improve their diagnosis, prognostic stratification and therapeutic outcome. We intend to focus on acute and chronic lymphoid leukemias (ALL and CLL), classic Hodgkin lymphoma (chl), peripheral T-cell lymphomas, including the not otherwise specified form (PTCL/NOS), angioimmunoblastic lymphoma (AITL) and anaplastic large cell lymphoma (ALCL) by exploring the following hypotheses: Codice Riferimento: 10007 Page 48 of 154
1) We hypothesize that pharmacological targeting of the BCR-ABL, ALK and PDGFRA/B oncogenic tyrosine kinases will diminish chemotherapy-induced toxicity and improve survival in patients with BCR-ABL + ALL, ALK + ALCL, PDGFR + PTCL-NOS and AITL, respectively. Such hypothesis will be tested in animal models and in phase I-II clinical trials. 2) We hypothesize that: i) the discovery by whole exome sequencing of novel key pathogenetic mutations in ALL, CLL, chl and PTCL will improve the molecular classification, diagnosis and prognostication of these lymphoid neoplasms through the development of specific immunohistochemical and/or molecular assays; and ii) newly discovered mutations can be translated into molecular targets by assessing their in vivo oncogenic potential and their susceptibility to a molecularly targeted therapy in appropriate mouse models and then in phase I-II clinical trials. Task 1: Pharmacological targeting of oncogenic TKs in selected lymphoid neoplasms. In spite of the continuous development of new tools for the treatment of lymphoid tumors, several neoplasms are still incurable, such as BCR-ABL+ ALL, PTCL-NOS and AITL. In addition, the toxicity of regimens effective on neoplasms like ALK+ ALCL remains high. Preliminary data generated by our consortium indicate that these tumors can be sensitive to TKI because of the constitutional activation of kinases through either specific chromosomal aberrations (BCR-ABL and ALK) or other mechanisms (PDGFR). This observation prompts to: 1) conclusively assess the efficacy of TKI, both alone and in combination with other drugs (vaccines, MoAb, HDAC inhibitors, etc), and 2) develop and test new schedules in phase I-II trials. ALL. We have pioneered the use of 1 st and 2 nd generation TKI as single agents for the first-line treatment of Ph+ ALL, including the elderly where Ph+ ALL accounts for ~50% of cases. We will expand this approach by: i) designing a complete non-chemotherapy strategy to control BCR/ABL + ALL with TKI alone as 1 st line induction treatment for all newly diagnosed patients (>18 yrs, no age limit), followed by a specific vaccination protocol or recently developed MoAb that trigger a T- cell response as maintenance/consolidation treatment; ii) rotating the available TKI with novel inhibitors active against the T315I mutation (the primary cause of resistance/relapse) currently under development; iii) incorporating - in a European effort - TKI in the treatment of T-ALL cases carrying ABL1 overexpression. ALK + ALCL. The genomic or pharmacological ablation of ALK signaling leads to cell death, corroborating the notion that ALK is a valid/specific target molecule. Aiming at identifying innovative treatments, we will: i) evaluate the ALK kinase inhibitor CEP-28122 (developed by Cephalon Inc in collaboration with us) as single frontline approach and/or in association with chemotherapy; ii) test whether tumor debulking with an ALK inhibitor may allow the synergistic success of immune-based therapies (i.e. vaccinations against ALK, antibody-based therapies specific for tumor and/or host targets, e.g. humanized anti-ccr4 MoAb) and/or the development of efficacious host immune-responses against ALK determinants; iii) evaluate the role of maintenance with CEP-28122 in ALCL patients in CR following conventional therapies (if chronic administration will prove safe in the pre-clinical setting). PTCL/NOS and AITL. We will: i) test the efficacy of TKI - as single agents or in combination with HDACi, MoAbs or chemotherapy - against PDGFRA + PTCL/NOS and AITL; ii) test the efficacy of anti-vegf compounds as single agents or in combination with chemotherapy against AITL. Codice Riferimento: 10007 Page 49 of 154
To accomplish the above goals we will need to generate pre-clinical models in which to test currently unexplored combination therapies. A new frontier in translational biomedical research is represented by highly immunocompromized NOG animals and/or humanized mice. These models are necessary, in particular, to evaluate immuno-based strategies (e.g. MoAbs, vaccinations), to study tumors lacking informative genetic models and to assess the relationship between tumor and host cells. Recently, we demonstrated the successful engraftment of lymphoma tissue samples and/or single cell preparations in NOD/Scid/IL2g-/- (NOG) mice and established: a) a large comprehensive tissue bank of viable cryopreserved samples embracing Italian centers cooperating with several European countries, US and Canada; b) a mouse colony of severely immunocompromized mice (NOD/Scid and NOG). We are thus in the position of systematically and reproducibly generating primary ALL and PTCLs tumor-grafts (including PTCL/NOS and ALCL) to test known and novel TKI alone and in combination with chemotherapy and/or other emerging tailored therapeutics. All available samples, as well as prospectively banked samples, will be implanted. Tumor growth will be monitored over time (MRI, PET, sonography, etc) and the reliability of each tumor-graft will be tested versus primary and correspondent tumor-grafts by GEP, array-cgh, IHC and FISH. From a clinical standpoint, the proponents have already obtained notable results with the use of specific inhibitors (non-chemotherapy concept) in various lymphoid disorders. Accordingly, it is planned that a number of targeted phase I-II studies will be designed and activated in the first 3 years of the project after appropriate pre-clinical testing. Task 2: Discovery of new/driving mutations in lymphoid neoplasms and their translation into molecular targets. chl. chl represents the third most common lymphoma. It affects mostly young patients, thus being aggravated by a significant burden of chemo/radiotherapy-induced late toxicities. chl is unique among all B-cell lymphomas due to its almost complete loss of B-cell identity and extreme rarity of neoplastic cells in the tumor. Although aberrant activation of oncogenic pathways such as NF-kB and JAKs-STATs is common in chl, no unifying genetic lesion has so far been identified that can comprehensively explain its dramatic cell phenotype reprogramming. Whole exome sequencing has the ground-breaking potential to: i) unravel the founding genetic lesion(s) of chl; ii) use this information to improve the differential diagnosis between chl and its mimics that require a different therapeutic approach (e.g. ALK-negative ALCL and primary mediastinal B-cell lymphoma) through the development of specific immunohistochemical and/or molecular assays; iii) allow for the first time the generation of mouse models suitable for disease understanding and testing of molecularly-targeted therapies; iv) transfer these targeted therapeutics to the clinic. Owing to the rarity of neoplastic cells in affected lymph nodes, whole exome sequencing will be performed in 6-8 well characterized chl cell lines. This approach precludes sequencing of the nonneoplastic cell counterpart as control. However, as chief guiding criterion to select pathogenetically relevant mutation(s) for validation in primary chl material, we will require that they be highly recurrent (i.e. occurring in all or most of the several chl lines) and, to some extent, affect biologically interesting genes. ALL. ALL remains a largely unmet medical need, particularly in adults, though the encouraging results obtained in Ph+ ALL with TKI strongly indicate that genetics-driven strategies are today a primary challenge that must be addressed. Different endpoints will be pursued. We will: i) determine and compare the underlying genetic profile in different molecular subgroups of B- and T- lineage ALL; ii) focus on ALLs without known genetic lesions; in this preponderant subgroup the identification of new genetic abnormalities would bear important diagnostic, prognostic and therapeutic implications, as well as new MRD monitoring possibilities; iii) evaluate possible differences in the frequency of genetic lesions according to age: children, adults, elderly; iv) Codice Riferimento: 10007 Page 50 of 154
clinically validate the prognostic implications of the information gathered above; v) use specific mouse models to test the transforming potential of newly identified lesions; vi) develop and validate novel therapeutic targets pre-clinically and through phase I-II clinical trials. CLL. In the era of highly effective combination therapies, two major clinical issues in CLL remain chemorefractoriness and transformation into aggressive lymphoma (Richter syndrome). In fact, the molecular basis of chemorefractoriness is not fully explained by the presence of TP53 disruption. Also, the molecular mechanisms associated with and predicting for CLL transformation to aggressive lymphoma are currently unknown and may unravel novel therapeutic targets. Whole exome sequencing will be utilized in particular in: i) CLLs devoid of the most common genetic lesions (i.e. +12, del13q14, del11q, del17p13), in order to identify the molecular alterations that drive these cases and possibly provide suitable therapeutic targets; ii) different clinical categories of CLL patients - at presentation, with documented long-lived stable disease, at first progression and at relapse - to unravel the genetic mechanisms underlying such markedly different clinical scenarios; iii) patients who are alkylator/fludarabine-refractory independent of TP53 alterations, to identify (and possibly overcome) unknown mechanisms of chemorefractoriness; iv) sequential samples of CLL and Richter syndrome (the sample repository, the largest world-wide, is already available) to identify the molecular events causing transformation from CLL to aggressive lymphoma. In view of the pronounced difference in CLL prevalence between the Western hemisphere (~30% of leukemias) and Asian countries (2-5%), a collaborative project will compare the genetic profile of CLL cases from our country and from Japan. PTCLs. PTCLs are relatively rare lymphomas characterized by aggressive behavior and dismal prognosis. Differently from the commonest B-cell lymphomas, PTCLs do not present with recurrent genomic aberrations, with the exception of ALK+ ALCL. By applying whole exome sequencing to a panel of PTCL/NOS, AITL and ALCL (ALK+ and ALK-) we expect to: i) unravel the founding genetic lesion(s) of PTCL/NOS, AITL and ALK- ALCL, and identify previously uncovered genetic defects in ALK+ ALCL; ii) identify novel diagnostic subgroups of tumors, characterized by different genomic aberrations, thus improving the often very difficult differential diagnosis in the field of ALK- PTCLs; iii) verify their potential prognostic relevance within a clinical setting; and iv) identify novel potential therapeutic targets to be validated in pre-clinical and clinical models. Whole exome sequencing is particularly challenging in PTCLs because of the often abundant reactive cellular infiltrate present in the tumor biopsies. Therefore, samples will be pre-selected for having at least 70% neoplastic cells and will be sequenced multiple times to ensure adequate coverage of all alleles (including the neoplastic ones) that are present in the biopsy. The next-generation sequencing aspect of this program will be investigated using a high throughput sequencing facility equipped with a Roche 454 Genome Sequencer FLX Instrument (Titanium Series) recently established at the Istituto Superiore di Sanità, Rome. The facility will be available to all partners to perform next-generation sequencing of the whole exome, which is more costeffective than sequencing the whole genome. Data management at the ISS facility will include the development of the computational methodology to decipher the network of genetic, transcriptional and functional interaction in lymphoid malignancy. Notably, sequencing data will be fully integrated with the currently utilized genetic work-up that already includes gene expression profiling (GEP) and SNP array. To develop the translational aspect of the program we will use: i) suitable animal models, including the xenografted or humanized ones described within Task 1, as well as (conditional) transgenic/knock-in or knock-out mice, to dissect in vivo the oncogenic function of the newly discovered mutations and to test their susceptibility to a molecularly targeted therapy in a preclinical setting; ii) the clinical trial facilities mentioned below. Codice Riferimento: 10007 Page 51 of 154
Program feasibility The outstanding goals of this coordinated program can only be achieved through a multiinstitutional framework. Most of the required key expertise and facilities are located in different groups throughout Italy and will represent the common platforms available to all groups to develop the hypotheses driving this proposal: tissue and cell banking (the largest collection of archival material in Italy, as well as of primary prospective samples) and hematopathology (Pileri, Bologna; Falini, Perugia; Inghirami, Turin; Gaidano, Novara; Foà, Rome), next-generation sequencing and bioinformatics (Belardelli, Rome), mouse modeling (Inghirami/Forni, Turin), coordination of phase I-II clinical trials (Foà, Rome). In this regard, a unique asset of this multi-institutional proposal strongly supporting its transferability, within the duration of the project, to the clinical setting is the prominent power in sample collection and patient availability afforded by the center in Rome, the largest in Italy, and by the cooperative groups GIMEMA, Intergruppo Italiano Linfomi-IIL and EWALL (European Working group for ALL), of which the PI (Foà) and some of the Group Leaders (Falini, Pileri and Gaidano) are prominent coordinators/members. Over many years, the GIMEMA and IIL have coordinated clinical protocols for acute and chronic leukemias and lymphomas in Italy (and Europe), and have also set up a framework for a central sample handling of patients enrolled the different trials. References Choi M et al. PNAS 2009 Oct 27. Weiss LA et al. Nature 2009;461:802-8. Martinelli G et al. J Clin Oncol 2009;27:5202-7. Rossi D et al. Clin Cancer Res 2009;15:4415-22. Kuppers R. Nat Rev Cancer 2009;9:15-27. Ley TJ et al. Nature. 2008;456:66-72. Swerdlow SH et al. WHO Press, 2008; pp 1-439. Vignetti M et al. Blood 2007;109:3676-8. Piccaluga PP et al. J Clin Invest 2007;117:823-34. Piva R et al. J Clin Invest 2006;116:3171-82. Codice Riferimento: 10007 Page 52 of 154
Foà Roberto [PI] ABSTRACT Principal Investigator's Full Name Professor Foà Roberto Institution and City Università di Roma "La Sapienza" Proposal Title Genetics-driven targeted management of lymphoid malignancies Area Keywords Leukaemia; Lymphomas; Diagnosis; Prognosis; Target therapy Abstract in the next page Codice Riferimento: 10007 Page 53 of 154
Foà Roberto [PI] Abstract The diagnosis, prognosis and management of acute and chronic hematologic malignancies has witnessed major changes over the years. This has stemmed from our progress in knowledge and better understanding of the underlying genetic lesions and is reflected by the fact that the recent 2008 WHO classification of lympho-hemopoietic neoplasms is increasingly genetically-oriented. All-trans-retinoic acid (ATRA) in adults and children with acute promyelocytic leukemia and tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia represent illuminating examples of molecularly-driven targeted therapy that have changed the natural course of these diseases. Such scenario can now be extended to lymphoid malignancies, which today represent the third most frequent cancer and are expected to become the first within 20 years. Our project will focus on acute lymphoid leukemia (ALL), the most frequent cancer in childhood, chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults and elderly, classical Hodgkin lymphoma (chl), the most common lymphoma in young adults, and peripheral T-cell lymphomas (PTCL), a group of disorders frequently harboring a very poor prognosis. The strongly mechanism-based endpoint of the proposal is to broaden the genetics-driven targeted management of lymphoid malignancies by designing innovative TKI-based therapeutic strategies, discovering new driving genetic mechanisms and exploiting the latter to improve diagnosis, prognostic stratification and therapeutic outcome. The aims of the project are more specifically the following: 1) Innovative pharmacological targeting of known oncogenic TKs. This will be pursued: a) in BCR/ABL+ ALL, by designing nonchemotherapy phase I-II clinical trials based on the use of available TKI, in combination with monoclonal antibodies and/or new TKI in rotating schedules to avoid selection of counter-acting mutations; b) in ALK + anaplastic large cell lymphoma (ALCL) and PDGFR + PTCL-NOS (not otherwise specified) in both preclinical and clinical models, based upon preliminary in vitro and exvivo results indicating a response to these compounds. The final goal is to improve overall survival, spare toxicity and hospitalization, and to provide valid alternatives to current chemotherapeutic options, also in the elderly. 2) Discovery of new driving mutations in lymphoid neoplasms and their clinical translation. Whole exome sequencing (WES) will be utilized to unravel yet unknown genetic hits that may play a pivotal role in malignant transformation of ALL, CLL, chl and PTCL, with particular focus on specific clinically-based questions. The biological and clinical significance of the newly identified mutations will be tested in large patients series and in animal models. The final goal is to improve the molecular classification, diagnosis and prognostication of these neoplasms through: a) the development of specific immunohistochemical and/or molecular assays; b) the correlation with available clinical and molecular features and outcome, ultimately leading to an early identification of patients who would fail treatment; c) the discovery of new potential therapeutic targets to be tested at the pre-clinical level, including the mouse model. The strength of the project lays also in the proposing Consortium, that includes internationally recognized partners with long-standing cooperation, large tissue banks and clinical trial networks, as well as innovative technological resources. Codice Riferimento: 10007 Page 54 of 154
PROPOSAL MAIN BODY Foà Roberto [PI] Genetics-driven targeted management of lymphoid malignancies Keywords: Leukemias, Lymphomas, Diagnosis, Prognosis, Target therapy General background Lymphoid malignancies are among the most frequent neoplasms, with acute lymphoid leukemia (ALL) being the commonest malignancy in children, classical Hodgkin lymphoma (chl) the most common lymphoma in young adults and chronic lymphocytic leukemia (CLL) the most frequent leukemia in adults and in the elderly. Lymphoid neoplasms represent nowadays the third cancer in terms of frequency, but the WHO estimates that in about 20 years they will be the first. Lymphoid disorders also include orphan diseases, e.g. peripheral T-cell lymphomas (PTCL) and anaplastic large cell lymphoma (ALCL) for which conventional treatment is associated with a poor prognosis (PTCL) and/or with significant toxicity (ALCL) considering the patients median age. In a state-of-the-art hematologic setting, the laboratory and the clinic concur to provide a rapid and accurate diagnostic work-up, differential diagnosis among akin disorders, reliable prognostic stratification and minimal residual disease (MRD) monitoring, suggestion of targeted therapies, integration of the latter in the clinical management of patients and, ultimately, an improved quality of life and overall survival. Breakthroughs in the treatment of hematologic neoplasms have stemmed from the identification of their underlying driving genetic lesion(s). Illuminating examples are represented by acute promyelocytic leukemia (APL) and chronic myeloid leukemia (CML). In APL, this has led to the clinical use of all-trans-retinoic acid (ATRA), that bypasses the differentiation block of the leukemic cells. Similarly, in CML the genetics-driven design of tyrosine kinase inhibitors (TKIs) has changed the natural history, management and outcome of this disease. The use of TKIs has recently extended to Ph+ ALL that shares with CML the same driving genetic lesion. These examples highlight the importance of a genetics-driven molecular understanding of cancer and unequivocally document the feasibility in hematology of bench-bedside translation and its profound clinical impact, in terms both of diagnostic classification, prognosis and treatment. To make this progress possible, a complex and highly skillful network is required that assembles diverse laboratories and clinical expertise, coupled to state-of-the-art as well as new technologies, from diagnostic hematopathology and tissue banking to next-generation sequencing, molecular biology dissection of oncogenic pathways and clinical testing of new drugs, in addition to operational cooperative networks. Overall aims of the program The unifying and strongly mechanism-based concept of the current proposal is to broaden the genetics driven-targeted management of lymphoid malignancies by designing innovative TKI-based therapeutic strategies, by discovering new driving genetic mechanisms and by exploiting the latter to improve diagnosis, prognostic stratification and therapeutic outcome. We intend to focus on acute and chronic lymphoid leukemias (ALL and CLL), chl, and PTCL, including the not otherwise specified form (PTCL/NOS), angioimmunoblastic lymphoma (AITL) and ALCL. The Consortium is represented by internationally recognized experts in the molecular, diagnostic and clinical management of such malignancies. RF, SP and BF are part of the steering committee for the WHO classification of hemopoietic neoplasms. In line with this, they work in (and were responsible of the set-up of) institutions that are referral centers for virtually all leukemias and lymphomas in Italy. Two major Tasks will be addressed. Codice Riferimento: 10007 Page 55 of 154
Task 1: Pharmacological targeting of the BCR-ABL, ALK and PDGFRA/B oncogenic TK. This strategy will be pursued in order to diminish chemotherapy-induced toxicity and improve survival in patients with BCR-ABL + ALL, ALK + ALCL, PDGFR + PTCL-NOS, respectively. Preliminary data generated by our Consortium indicate that also these PTCL tumors can be sensitive to TKI (see below). For BCR/ABL+ ALL this hypothesis will be tested clinically, for ALK+ ALCL and PDGFR + PTCL-NOS in both preclinical and clinical models. Given that resistance and toxicity might still occur, discovery of new, driving genetic alterations (pursued in Task 2 for improving classification and prognostication) is instrumental to provide additional druggable targets in the future. Task 2. Discovery of new/driving mutations in lymphoid neoplasms and translation into molecular targets. The discovery by whole exome sequencing (WES) of novel key genetic mutations in ALL, CLL, chl and PTCL will improve the molecular classification, diagnosis and prognostication of these lymphoid neoplasms through the development of specific immunohistochemical and/or molecular assays. The experimental strategy and methodology concerning WES and subsequent validation, being conceptually shared by each disease, is outlined in a separate section ( WES and its validation ). The two general tasks will be addressed in specific disease models, namely: i) ALL: Sub-tasks 1A and 2A; Milestones 1-3 ii) PTCL: Sub-tasks 1B and 2B; Milestones 4-7 iii) CLL: Sub-task 2C; Milestones 8-11 iv) chl: Sub-task 2D; Milestones 12, 13 Given the strong disease-oriented nature of the project, the description of the research will be subdivided into sections corresponding to the specific diseases investigated (Table 1). According to this model, sub-tasks 1A and 1B refer to the overall Task 1 outlined above, while sub-tasks 2A, 2B, 2C and 2D refer to the overall Task 2 outlined above. Table 1. Organization of the project sub-tasks and milestones for each disease model. Disease model ALL Sub-task 1A Milestone 1. TKI-based phase I-II clinical trials Sub-task 2A Milestone 2. WES of ALL without known genetic abnormalities in different age cohorts and at different time points (onset and recurrence/resistance) Milestone 3. Clinico-pathological correlations and translation PTCL Sub-task 1B Milestone 4. Preclinical models Milestone 5. Clinical models Sub-task 2B Milestone 6. WES in PTCL Milestone 7. Validation of findings and clinical correlations CLL Sub-task 2C Milestone 8. Unraveling novel mechanisms of CLL chemorefractoriness Milestone 9. Identifying novel molecular markers of CLL clonal evolution Milestone 10. Unraveling the molecular basis of CLL transformation to RS Milestone 11. Pathogenicity and therapeutic exploitation of identified CLL mutations chl Sub-task 2D Milestone 12. Unraveling new driving genetic lesion(s) in chl Milestone 13. Clinical translation of the newly found genetic lesion(s) Codice Riferimento: 10007 Page 56 of 154
The overall organization within the Consortium, the task interconnections, the involvement of each unit in each disease model, as well as the team relationship are graphically illustrated in Fig. 1. Overall organization, Task interconnections and Team relationship Task 1: Development of innovative therapeutic approaches based on TK targeting in lymphoid neoplasms 4 PI 5 Phase I and II Clinical trails 1 4 5 Pre-clinical Tg and tumorgraft models A2- and NSG mice Tg mice New target drugs Task 2: Discovery of new driving mutations in lymphoid neoplasms and their clinical translation PI ALL PI 3 CLL 1 WES PTCL HD 2 chl 4 5 Functional validation HTP platforms and bioinformatics Fig. 1. Overall organization chart and interaction within the Consortium. Diagnostic and prognostic assay developement GIMEMA EWALL IIL Primary Human Cancers DNA/RNA, Tissue Banks 5 UniBo 4 UniTo PI UniRo Consortium 3 UniNo 2 UniPg 1 ISS Abbreviations: WES, whole exome sequencing; ALL, acute lymphoid leukemia; PTCL, peripheral T-cell lymphoma; CLL, chronic lymphocytic leukemia; chl, classical Hodgkin lymphoma; GIMEMA, Gruppo Italiano Malattie EMatologiche dell Adulto; EWALL, European Working Group for ALL; IIL, Intergruppo Italiano Linfomi; UniRo, Sapienza University of Rome; UniPg, University of Perugia; ISS, Istituto Superiore di Sanità (Rome); UniNo, University of Eastern Piedmont (Novara); UniTo, University of Torino; UniBo, University of Bologna. ALL Background and rationale ALL occurs at all ages, with a peak in childhood and a progressive increase in the elderly. In adults, ALL remains a largely unmet medical need (Vitale et al, 2006; Pui et al, 2008). Different genetic lesions can be found in about 50% of B-lineage ALL and are associated with a particularly poor prognosis (Mancini et al, 2005). The most frequent abnormality is represented by the BCR/ABL rearrangement, that accounts for 30-40% of adult ALL (Gleissner et al, 2002; Mancini et al, 2005) and for ~50% of elderly ALL (Burmeister et al, 2008). At all ages, BCR/ABL+ ALL is associated with a poor response to conventional chemotherapy regimens and an overall dismal prognosis (Moorman et al, 2007). New technologies, i.e. gene expression profiling and SNP array have allowed to define signatures associated with molecular aberrations and to identify novel patients subgroups (Mullighan et al, 2008 & 2009; Iacobucci et al, 2009). Although the understanding of the genetic profile of ALL has led to the utilization of TKIs in the management of BCR/ABL+ ALL (Druker BJ et al, 2001; Kurzrock et al, 2003; Talpaz M et al, 2006; Ottmann et al, 2007), resistance to conventional chemotherapy and to 1 st and 2 nd generation TKIs still represents a major clinical problem. In addition, the driving genetic lesions of BCR/ABLnegative ALLs are still largely unknown. Codice Riferimento: 10007 Page 57 of 154
Sub-task 1A: Development of innovative therapeutic approaches based on TK targeting Questions and objectives 1) Design of TKI-driven therapeutic protocols aimed at controlling the disease without chemotherapy and transplant procedures. 2) Applicability of such a strategy to the elderly (with no upper age limit), where the incidence of BCR/ABL+ cases is preponderant. Milestone 1: TKI-based phase I-II clinical trials (1 st to 4 th year) Experimental design The PI group has pioneered the efficacy of TKIs in BCR/ABL+ ALL, also in the elderly (Vignetti et al, 2007; Foà et al, 2008). We will expand this approach by: i) designing a complete nonchemotherapy strategy to control BCR/ABL+ ALL, based on the use of the 2 nd generation TKI dasatinib alone as induction treatment followed by the administration of a bispecific B-T MoAb (BiTE) as consolidation (Fig. 2); 2) rotating the available TKIs with novel inhibitors (e.g. the Aurora kinase) active against the T315I mutation (Moore AS et al, 2010), the primary cause of resistance to 1 st and 2 nd generations inhibitors, currently under development (Fig. 2). Based on the current knowledge and operational framework, it is expected that the initial trial can be designed within the 1 st year of the project. NON-CHEMOTHERAPY TREATMENT OF BCR/ABL+ ALL Steroids pre-treatment Induction: Dasatinib + Steroids Response evaluation: day +85 MRD- MRD+ Fig. 2. Design of a nonchemotherapy protocol for BCR/ABL+ ALL based on the use of a TKI and of a bispecific (anti- B/anti-T) MoAb or of a TKI directed against the T315I mutation. Stem cell mobilization BiTE MoAb or anti-t315i TKI Dasatinib maintenance MRD monitoring Dasatinib maintenance Methodological approach and feasibility The GIMEMA (Gruppo Italiano Malattie EMatologiche dell Adulto) and EWALL (European Working group for ALL) networks guarantee an optimal accrual of patients throughout Italy and Europe, and the necessary biologic network. Sample size analysis before patients accrual will be performed: an enrolment of ~40 cases for each regimen is expected throughout the above national and international frameworks. Significance and impact of expected findings This strategy will ultimately lead to: i) verify the possibility of controlling BCR/ABL+ ALL, the most lethal leukemia, without chemotherapy; ii) effectively treat also elderly patients with this disease, who would otherwise receive only palliation therapy; ii) spare the significant toxicity and Codice Riferimento: 10007 Page 58 of 154
need for hospitalization associated to conventional and myeloablative chemotherapy in all age groups, thus improving compliance and quality of life. Sub-task 2A: Discovery of new driving mutations and their clinical translation. Questions and objectives 1) Discovery of unrevealed genomic lesions involved in ALLs not carrying known genetic abnormalities in various age groups. 2) Definition of their diagnostic and prognostic impact. 3) Identification of mutations associated to disease recurrence/resistance. Milestone 2: WES of ALL without known genetic abnormalities in different age cohorts and at different time-points (onset and recurrence/resistance) (1 st to 4 th year) Experimental design WES will be conducted in B- and T-lineage ALL without known genetic aberrations. Given the different prognosis of B-lineage ALL according to age, patients belonging to various age cohorts (children, adolescents, young adults, adults, elderly) will be analyzed separately. In addition, samples taken at disease onset will be compared by WES to those taken at relapse from the same patients, to identify genetic events associated with chemoresistance/relapse. Methodological approach and feasibility Biological material (i.e. high quality genomic DNA, viable cells frozen cryopreserved in DMSO as well as total RNA) is available for various disease time-points at the PI Center. In fact, the Institution in Rome coordinates several protocols for the management of adult ALL through the GIMEMA network, ensuring adequately sized cohorts of patients. For the pediatric cases, samples will be available through the PI center and through the AIEOP (Associazione Italiana di Ematologia ed Oncologia Pediatrica). Milestone 3. Clinico-pathological correlations and translation (5 th year) Experimental design New mutations discovered in Milestone 2 will be correlated with clinical (i.e. age, features at onset of disease and response to therapy) and biological variables (immunophenotypic, molecular analyses and gene expression profiling) to test their impact on clinical outcome and on the molecular features of the leukemic cells. Methodological approach and feasibility The availability of a large clinical database will allow to evaluate retrospectively, and possibly prospectively, the predictive power of the lesions identified and will be fully integrated with molecular analyses performed on all cases to evaluate the presence of recurrent molecular fusion transcripts. Moreover, for most patients, gene expression profiling data are also available through our Affymetrix platform. The GIMEMA Data Center has been collecting clinical and biological information for all ALL patients enrolled in the national protocols for over 12 years; furthermore, biostatisticians devoted to this type of statistical analyses are available. Significance and impact of expected findings In the short-term, the molecular knowledge obtained by WES will improve patients classification and prognostic stratification, and will allow a genetics- and biologically-based administration of already available therapeutic tools, thus avoiding under or overtreatment. In the long-term, such molecular knowledge will be instrumental to the design of new targeted therapies. PTCL Background and rationale Codice Riferimento: 10007 Page 59 of 154
PTCLs account for approximately 12% of lymphoid neoplasms (Swerdlow et al, 2008). Four main subtypes represent 70-75% of cases (Vose et al, 2009): PTCL not otherwise specified (PTCL/NOS), angioimmunoblastic T-cell lymphoma (AITL), ALK + ALCL and ALK - ALCL. Although the new WHO Classification represents a step forward in the definition of these tumors (Swerdlow et al, 2008), several issues are still open. For instance, PTCL/NOS is by definition a basket category, where heterogeneous morphologic and phenotypic patterns are included until they can be better classified, while ALK - ALCL has a provisional status, needing definitive clinico-pathological confirmation. These uncertainties affect the daily clinical practice by making the diagnosis of these tumors laborious and not easily reproducible (Vose et al, 2009). Clinically, PTCLs are among the most aggressive non-hodgkin lymphomas (NHL): except for ALK + ALCL, response to conventional chemotherapy is frustrating, with relapse-free and overall survival rates at five years of 26% and 20%, respectively (Swerdlow et al, 2008). The molecular bases of PTCLs remain elusive. Several studies have focused on their molecular profiling (Piccaluga et al, 2007; Martinez- Delgado et al, 2004; Piccaluga et al, 2005; Piccaluga et al, 2007; de Leval L et al, 2007; Iqbal et al, 2009; Hartmann et al, 2010; Piva et al, 2010) and postulated the existence of different subtypes characterized by a distinct cellular derivation (Piccaluga et al, 2007; Piccaluga et al, 2007b; de Leval et al, 2007). ALK + ALCL represents an informative model showing that the deregulated activation of the ALK gene is indispensable for the maintenance of the neoplastic phenotype (Piva et al, 2010). In addition, recent studies have identified novel potential therapeutic targets including TKIs, histone deacetylase inhibitors (HDACi) and proteasome inhibitors (PrI) (Piccaluga et al, 2007; Martinez-Delgado et al, 2004; Piccaluga et al, 2005; Piccaluga et al, 2007; de Leval L et al, 2007; Huang et al, 2009). TKs aberrant activation seems to be a possible common event in the pathogenesis of PTCLs (Piccaluga et al, 2007; Martinez-Delgado et al, 2004; Piccaluga et al, 2007; de Leval L et al, 2007; Iqbal et al, 2009; Piva et al, 2010). In particular, PDGFRα is over-expressed at both the gene and protein level, where it is consistently phosphorylated. Accordingly, TKIs appear highly effective against PTCL cells ex vivo (Piccaluga et al, 2007, Huang et al, 2009). Finally, specific ALK TKIs have shown therapeutic efficacy in mouse and human cell-based in vivo models (Piva et al, 2006, Wan et al, 2006; Galkin et al, 2007; Chiarle et al, 2008; Du et al, 2009). Sub-task 1B: Development of innovative therapeutic approaches based on TK targeting Questions and objectives 1) Development of innovative experimental models to validate known and newly identified molecular therapeutic targets. 2) Definition of the mechanisms sustaining TKs aberrant activation, to identify all involved genes/pathways and to characterize: a) possible mechanisms of TKI resistance, and b) second generation TKIs. 3) Design and implementation of phase I-II clinical trials to assess the benefit of targeted/selective drugs. Milestone 4: Preclinical models (1 st to 2 nd year) Experimental design Preclinical chemotherapeutic protocols, combining conventional compounds and TKIs of 1 st and 2 nd generation, will be evaluated using mouse models (Transgenic and immunodeficient [NSG] mice) tailored for PTCL of both the NOS and ALK + ALCL type, to select the most appropriate molecules and/or combinations. In this respect, the sensitivity of PTCL/NOS cells to TKIs has so far been assessed only ex vivo, while the ALK signaling ablation has been tested on human ALCL cell lines (including animal models), but not in primary cases. As to the latter point, we have recently generated a set of tumor-grafts of ALK + primary ALCL in NOD/Scid/IL2 -/- (NSG) which will be used to assess the therapeutic efficacy of a battery of ALK TKIs and to study the possible occurrence of drug-related mechanisms of resistance. The molecular characterization of primary and tumor-graft neoplasms should allow the discovery and/or validation of pathogenetic lesions. Codice Riferimento: 10007 Page 60 of 154
These studies constitute the necessary pre-requisite for the discovery of novel target molecules and for the definition of the mechanisms responsible for acquired TKI resistance. Methodological approach and feasibility Tumor bearing mice will be screened using MRI; positive animals will be treated per os with escalating doses of different TKIs over time (7-14 days single or multiple rounds every 21 days). Response to therapy will be assessed using imaging approaches. Cell proliferation, apoptosis and systemic dissemination potential will then be analyzed in vivo by Luciferase assay (after lentiviral transduction with a bidirectional cassette Luc-CMV-EGFP) and in vitro by conventional proliferation, apoptosis and invasion assays on explanted tumors. The analysis of the concordance between primary tumors and tumor-grafts in terms of histology, gene alterations, mrna and protein expression profiles is mandatory to verify the suitability of in vivo mouse models for individual testing of responsiveness towards conventional or innovative therapeutic strategies. The correspondence and similarity between primary and tumor-grafts will be investigated by: (i) IHC and inverse antibody capture; (ii) SNP (500k card) analysis; FISH karyotyping; (iv) HTP phosphomapping; and (v) gene expression profiling. The definition of driver mutations will be eventually assessed using in vitro functional validation approaches. Milestone 5: Clinical models (2 nd to 4 th year) Experimental design Phase I-II clinical trials based on the administration of 1 st and 2 nd generation TKIs targeted to PDGFRs and ALK protein will be designed. The drugs will be given alone and in combination with conventional therapies (i.e. CHOP or CHOP-like regimens) (Vose et al, 2009) to assess the safety and efficacy of the proposed schedules. In particular, the TKIs (1 st and/or 2 nd generation) to be tested in the clinic will be chosen based upon the results obtained in the mouse model. The experimental schedules will be drafted accordingly and submitted to the approval of the Ethical Committee. Methodological approach and feasibility The endpoints of the trials will be assessment of safety, complete remission rate and overall, disease-free and progression-free survival. Toxicity will be evaluated according to WHO criteria. Statistical tests will be performed by appropriate tools developed by the Intergruppo Italiano Linfomi (IIL) Data Center. Cases will be enrolled within the IIL network. An accrual of 50-80 cases per year is expected. Based on sample power analysis, such number suffices to answer the above mentioned questions. Significance and impact of expected findings A better understanding of the mechanisms sustaining sensitivity of PTCL cells to conventional and 2 nd generation TKIs, as well as the conduction of in vivo studies (including phase I-II clinical trials) based on such drugs, will provide alternatives to current therapeutic options that are known to be largely ineffective in the PTCL setting. Sub-task 2B: Discovery of new/driving mutations in lymphoid neoplasms and clinical translation Questions and objectives By applying WES to a panel of PTCL/NOS, AITL and ALCL (ALK+ and ALK-) we expect to: 1) Unravel the founding genetic lesion(s) of PTCL/NOS, AITL and ALK- ALCL, and identify mutations associated with chemo-refractoriness in ALK+ ALCL. 2) Identify novel diagnostic subgroups of tumors, characterized by different genetic aberrations, thus improving the often very difficult differential diagnosis in the field of ALK- PTCLs. 3) Detect novel therapeutic targets to be tested in in vitro, ex vivo and in vivo conditions including the mouse model. Codice Riferimento: 10007 Page 61 of 154
Milestone 6: WES in PTCL (1 st to 5 th year) Experimental design A series of 60 PTCLs with an amount of neoplastic cells exceeding 70% of the whole examined population and corresponding to 30 NOS cases (of the central memory, cytotoxic and follicular T- helper phenotypes), 10 AITLs and 20 ALCLs (10 ALK + both chemo-sensitive and resistant and 10 ALK - ) will be used for WES. Methodological approach and feasibility Collection of 20 cases per year with fresh pathological and normal tissue samples is expected, based on the historical background of the proponents and IIL network. WES will be performed according to the methodological approach defined by the Consortium. Moreover, in SP and GI registries there are over 50 cryopreserved PTCL and 30 ALCL samples that guarantee the execution of this task. Milestone 7: Validation of findings and clinical correlations (1 st to 5 th year) Experimental design New mutations, validated as indicated in the section of WES and its applications, will serve as the basis for the development of specific molecular or immunohistochemical tests applicable for routine diagnosis. Methodological approach and feasibility In synthesis, data validation of individual lesions detected by WES will be first performed by conventional Sanger sequencing targeting the coding region and splicing sites of selected genes, followed by additional PCR, FISH on key-genes/gene-products assays in an independent series. Genetic (including gene expression profiling, with HumanWG-6BeadChips v2.0 - Illumina or HG- U133 Plus 2.0 arrays - Affymetrix, and SNP with array GeneChip Human Mapping 250K NspI - Affymetrix) and immunohistochemical tests with ad hoc antibodies will be executed to determine the relevance/pathogenicity of selected aberrations based on bioinformatics data and literature findings. Finally, the assessment of the prognostic and diagnostic power of findings along with the development of preclinical models will be carried out. For this purpose, a historical cohort of 200 PTCLs with complete clinical information is available. This along with the existing facilities and skills guarantees the feasibility of this part of the project. Significance and impact of expected findings The identification of yet unidentified recurrent genomic lesions will help to: a) better understand the pathogenesis of PTCL/NOS, AITL, ALK+ ALCL and ALK- ALCL; b) identify novel diagnostic and prognostic tools leading to an easier categorization of PTCLs and a better stratification of patients; c) design innovative targeted therapies to be tested in ex vivo and in vivo models. CLL Background and rationale CLL is the commonest leukemia in our hemisphere (Moreno et al, 2008). Unmet clinical needs still include: i) chemorefractoriness to fludarabine, a mainstay drug of CLL treatment; ii) clonal evolution, leading to progressively more aggressive and chemorefractory phenotypes; iii) transformation to aggressive lymphoma (Richter syndrome, RS). These pitfalls account for 80% of deaths in patients requiring treatment. Chemorefractoriness due to TP53 inactivation is restricted to 30-40% of CLL not responsive to fludarabine (Zenz et al, 2008, 2009; Dicker et al, 2009, Malcikova et al, 2009; Rossi et al, 2009e), indicating that other molecular mechanisms account for chemorefractoriness in >50% CLL failing therapy. Clonal evolution drives the stepwise progression from clinically stable CLL not requiring treatment to progressive disease with poor prognosis. Understanding the molecular basis of clonal evolution may identify at diagnosis patients at risk of clinical progression and reassure patients who, despite being assigned a leukemia diagnosis, are projected to remain clinically stable. A precise definition of the risk of clonal evolution has become Codice Riferimento: 10007 Page 62 of 154
even more challenging since the identification of monoclonal B-cell lymphocytosis (MBL), a condition highly prevalent in the general population that may precede CLL development (Rawstron et al, 2008; Landgren et al, 2009; Shanafelt et al, 2009; Rossi et al. 2009d). RS represents transformation from CLL to aggressive lymphoma (Tsimberidou et al, 2005, 2006; Rossi et al., 2009) and is predicted by recently disclosed biological risk factors (Aydin et al, 2008; Rossi et al, 2008, 2009b, 2009c). Once RS has developed, outcome is extremely somber, with survivals <12 months (Tsimberidou et al, 2005; Rossi et al, 2009). Because most RS are intrinsically chemorefractory and not candidate to bone marrow transplantation, targeted therapies are highly desirable. This approach, however, is hampered by lack of molecular knowledge in RS. Sub-task 2C: Discovery of new driving mutations and their clinical translation Questions and objectives 1) Understand the mechanisms of chemorefractoriness in TP53 wild type CLL, in order to develop molecular predictors of fludarabine failure. 2) Understand the genetic determinants of clonal evolution, in order to develop clinical grade molecular markers that, at diagnosis, distinguish highly stable CLL from CLL prone to progress. 3) Understand the molecular basis of CLL transformation to aggressive lymphoma/rs, in order to develop novel tools for improving diagnosis and prognosis, and provide rational therapeutic targets. Milestone 8. Unraveling novel mechanisms of CLL chemorefractoriness (1 st to 2 nd year) Experimental plan: WES will be applied to cases refractory to fludarabine and carrying wild type TP53 alleles. For comparison, fludarabine-sensitive cases will also be investigated. Newly identified mutations associated with chemorefractoriness will be tested in large clinical series in order to develop molecular predictors that, at diagnosis, might herald therapy failure. Methodology and feasibility. Samples of fludarabine-refractory (n=10) and fludarabine-sensitive (n=10) CLL are already available for WES studies. Germline DNA will be obtained from buccal swabs, saliva or urine samples. A large CLL consecutive series (n=505), as well as an independent prospective series from a GIMEMA clinical trial (n=300), will be tested for newly identified mutations by conventional mutation screening assays in order to: i) characterize the frequency and recurrency of the genetic lesion; ii) validate the association with chemorefractoriness. The consecutive series is already available; the GIMEMA trial series will be collected during the first year of the study. Feasibility is based on: i) a large CLL network providing tumor samples; ii) availability of the WES facility within the Consortium (Unit led by FB). Milestone 9. Identifying novel molecular markers of CLL and MBL progression, and clonal evolution (2 nd to 3 rd year) Experimental plan: In order to characterize the molecular markers associated with CLL progression, WES will be applied to long-term stable CLL (n=10) and rapidly progressive CLL (n=10) with paired germline DNA. Conventional mutation screening assays will be applied to a consecutive CLL cohort in order to: i) define the frequency of the newly identified mutations; ii) define their impact on clinical endpoints, including time to first treatment and overall survival. In addition, WES will be applied to sequential paired samples of progressive MBL and CLL (n=20) in order to identify genetic lesions acquired at each step of clonal evolution. Newly identified mutations associated with clonal evolution will be tested in large clinical series to develop molecular predictors of MBL and CLL clinical course. Methodology and feasibility. Samples of long-term stable and rapidly progressive CLL, as well as paired sequential samples of MBL and CLL, are already available for WES studies. MBL samples will be sorted to obtain a fraction of CD19+/CD5+ cells >90%. Clinical validation of mutations will be performed on large consecutive CLL and MBL series collected through the Consortium. Codice Riferimento: 10007 Page 63 of 154
Milestone 10. Unraveling the molecular basis of CLL transformation to RS (2 nd year) Experimental plan: WES will be applied to paired sequential samples of CLL and RS with diffuse large B-cell lymphoma (DLBCL) histology (n=10). Genes found to be mutated by WES will be subsequently tested by Sanger sequencing in an extended panel of RS in order to assess i) the frequency and recurrency of the genetic lesions; and ii) their impact on RS clinical course. In parallel, an ongoing collaborative project (R. Dalla-Favera, Institute of Cancer Genetics, Columbia University) dedicated to DLBCL arising de novo will allow exchange of novel sequences and reagents for comparative analysis of RS versus de novo DLBCL. Methodology and feasibility: A large fully characterized RS series (all DLBCL; n=153) is already available at one participating institution (Unit led by GG). Paired DNA from the CLL phase is also available in selected cases (n=52). Milestone 11. Pathogenicity and therapeutic exploitation of identified CLL mutations (4 th and 5 th year) Experimental plan: The pathogenicity of novel recurrent mutations identified in i) chemorefractory CLL, ii) clinically progressive MBL and CLL, and iii) RS, will be addressed by appropriate in vitro studies and animal models. Xenograft animal models will be used to target mutations by novel drugs. Methodology and feasibility: Methods will include in vitro transformation, chemosensitivity and sirna assays, and transgenic and xenograft animal models. Feasibility is documented by availability of this technology within the network (Units led by SAP and GI). Significance and impact of expected findings 1. Identification of novel chemorefractoriness predictors in CLL may translate into: i) establishment of new genetic markers that increase our ability of identifying a priori patients who will fail fludarabine and require alternative treatments; ii) discovery of new therapeutic targets in a setting of patients in which treatment options are currently narrow. 2. Unraveling the molecular determinants of MBL and CLL clonal evolution will allow to identify at diagnosis patients who will remain clinically stable versus patients destined to progress. 3. Understanding the molecular basis of CLL transformation to RS may identify: i) novel predictors of CLL evolution to RS and of RS outcome post-transformation; ii) pathogenetic mutations that may be targeted by molecular therapy. chl Background and rationale chl represents the second most common lymphoma in Western countries overall, and the most common in young people (Han et al, 2008). Although often curable, it is aggravated by chemo/radiotherapy-induced late toxicities (Ng et al, 2007). Moreover, a minor but sizable group of chl patients (~15-20%) does not respond to standard therapies and their upfront identification is a major unmet clinical need (Canellos et al, 2007). chl is unique among B-cell lymphomas because neoplastic cells (Hodgkin-Reed/Sternberg HRS - cells) are a minority (usually <5%). Moreover, although deriving in >95% of cases from germinal center-experienced B cells, HRS cells have lost the B-cell transcriptional program, while expressing several genes of other hematopoietic lineages (Küppers, 2009). A common molecular feature of chl is the aberrant activation of NF-kB and JAK-STAT pathways, which is thought to mainly provide anti-apoptotic/proliferative signals to HRS cells. This can be promoted by various types of genetic alterations (Küppers, 2009). However, these genetic alterations are not present in all patients nor are they specific for chl, being observed in other lymphoma entities. Moreover, no unifying genetic lesion(s) has (have) been so far identified that can comprehensively explain the unique phenotype reprogramming of chl. The latter may be Codice Riferimento: 10007 Page 64 of 154
linked to neoplastic transformation, since the HRS cell phenotype does not resemble any normal B- cell subset. Knowledge of such key genetic lesion(s) could be exploited to resolve the problematic diagnostic grey zone between chl and other aggressive lymphomas (Swerdlow et al, 2008). Sub-task 2D: Discovery of new/driving mutations and their clinical translation Questions and objectives 1) To find new driving genetic lesion(s) in chl, in particular those responsible for HRS cell phenotype reprogramming and those associated with chemo-refractoriness. 2) To exploit new genetic lesions clinically by developing genetics-based specific immunohistochemical and/or molecular assays for improving the differential diagnosis between chl and its mimics and for identifying chl patients not likely to respond to standard therapies. Milestone 12: Unraveling new driving genetic lesion(s) in chl (1 st to 3 rd year) Experimental design Whole exome capture followed by massively parallel sequencing is at present not technically feasible using the minute amounts of genomic DNA (at most a few nanograms) obtained by pooling single HRS cells microdissected from chl biopsies. Thus, we will analyze 8 well-characterized chl cell lines (L1236, L428, KMH2, HDLM2, UHO1, SUP-HD1, L540, L591 - mostly derived from end-stage, chemorefractory patients) and 10 selected primary tumor biopsies exceptionally rich in HRS cells from typical, therapy-responder patients. The genotyping of cultured rather than of primary neoplastic cells offers the advantage of dealing with a 100% pure tumor cell population, but has two potential drawbacks: i) detection of mutations intervened during in-vitro cell line establishment/culture; and ii) inability to analyze non-neoplastic cells from the same individual, to ascertain the somatic nature of the mutations. However, the pathogenetically driving mutations (occurring either in a single gene or in different genes involved in the same functional process) might be retained in cultured HRS cells and might be recurrent across the 8 chl cell lines. The recurrency criterion will also help to distinguish driving events from the passenger mutation load that may characterize the quite unstable HRS cell genome. The above drawbacks will be tackled by validating in primary HRS cells microdissected from chl biopsies the top-ranking clonal mutations that are either non-synonimous or disrupt splicing sites, and that will be prioritized based on: i) absence from the reference genome and the ever growing databases of polymorphic genetic variants; ii) recurrence rate across the chl lines (confirmed with conventional Sanger sequencing); iii) predicted functional consequence; and, to some extent, iv) the role of the affected gene(s). To verify the somatic versus germline nature of the mutations, also nonneoplastic cells will be microdissected and studied. Exome-sequencing will also be performed on whole sections of chl biopsies very rich (up to 40-50%) in HRS cells. Multiple sequencing rounds will be performed to reach the sufficiently deep coverage required for a reliable detection of mutated alleles among a majority of wild-type alleles. Non-neoplastic cells (e.g. peripheral blood granulocytes) from these patients will be exomesequenced as well for prioritizing mutations also according to their somatic nature before validation in typical chl biopsies. Methodology and feasibility Several dozens of frozen chl biopsies with clinical annotations (including cases very rich in HRS cells, as well as chemorefractory cases) are available for analysis in the tissue banks from our cooperative group. PALM laser microdissection-catapulting system and two-rounded PCR for single/few cells will be performed as we previously reported (Liso et al, 2006). Details on WES are given below (section WES and its validation ). Milestone 13: Clinical translation of the newly found genetic lesion(s) (3 rd to 5 th year) Experimental design Codice Riferimento: 10007 Page 65 of 154
Discovery of the key, chl-specific genetic lesion(s) will bear clinical relevance by helping in the distinction between chl from its mimics that require a different therapeutic approach. For example, some lymphomas have clinico-pathological features intermediate between chl and diffuse large B- cell lymphoma or primary mediastinal B-cell lymphoma (Swerdlow et al, 2008). Diagnostic uncertainty sometime arises also between tumor-cell-rich chl versus ALK-negative anaplastic large cell lymphoma. Moreover, comparison of the exome-wide mutation profile of chl cell lines (reflecting end-stage, chemorefractory patients) and primary HRS cells from typical therapyresponder patients could unravel genetic lesions associated with therapy failure, that were either present at disease onset or acquired at relapse. After validation in primary HRS cells isolated from typical chl biopsies of therapy-sensitive versus therapy-resistant patients by conventional Sanger sequencing, these mutations can be translated into prognostication tools. The development of a PCR-based assay detecting the chl-specific or the chemorefractoryassociated mutated gene(s) on whole sections will be actively pursued, but it might be applicable only to cases relatively rich in neoplastic cells. A more general approach is to raise MoAbs against the product(s) of mutated gene(s) identified in Milestone 1, that are either specific for the mutated versus wild-type protein or identify the mutated protein through a subcellular distribution different from the wild type. Methodology and feasibility MoAbs against fixative-resistant epitopes preserved in routine paraffin-embedded biopsies will be generated by the hybridoma technology facility well established at the Institute of Hematology, Perugia. This facility has produced numerous MoAbs used worldwide for diagnosing hematological malignancies (Falini et al, 2002). A large paraffin tissue bank of lymphomas of any type and of chl with clinical annotations is available in our cooperative group for antibody testing and validation. Significance and impact of expected findings WES of HRS cells has the following multifold ground-breaking potential in chl: i) discovery of the yet unknown, specific genetic lesion(s) of chl; ii) improved classification and thus improved therapeutic outcome of borderline cases with intermediate (chl versus NHLs) features; iii) improved prediction of standard therapy failure, and thus improved clinical management, of chl patients. WES and its validation The discovery phase of the project will rely on WES applied to samples collected from patients suffering from the above described lymphoid malignancies, to outline the genetic lesion map of tumor samples, primarily nonsynonymous, splice site or indel sequence variants. A general strategy will be applied to the relevant clinical questions described in detail for each disease and will consist of the following steps. 1) WES on a discovery panel of samples. In this explorative phase, no statistical hypothesis is postulated. For each clinical question, phenotypically homogeneous patients will be randomly chosen; samples will be collected from the malignant and the normal tissue control of each individual patient and/or from paired sequential phases of the disease. By enrolling 5 patients we are confident (probability >0.9) that at least 1/5 cases will carry a genetic lesion, which has a 40% prevalence in the study population. Similarly, by enrolling 10 patients, we will uncover also less frequent genetic lesions (prevalence of 20%). 2) Variants calling. The pipeline to be applied to the WES data for the mutation discovery will require that the genetic variations specific and unique for each individual genotype, as well as the known polymorphisms, will be filtered out. Candidate mutations will be further restricted by recurrence criteria within the same clinical group. Particular attention will be focused on the biological implication of the mutated gene(s) identified and on their documented involvement in other tumors. Codice Riferimento: 10007 Page 66 of 154
3) Extension panel. Direct Sanger sequencing will be used to confirm the mutations found by WES first in the discovery panel samples, and then in an independent and larger data set of individuals. In the latter data set, genes found by WES to carry one mutation in a particular exon will be screened for additional mutations in other coding exons and splicing sites, in order to have a more comprehensive estimate of the recurrence rate of any putative pathogenetic mutation. A test for comparing two proportions will be applied and the sample size will be calculated accordingly, by assuming a type 1 error rate of 5% and a statistical power of 80%. For example, we estimate that 37 subjects per group will be needed to confirm that 20% of the subjects showing the selected phenotype carry the genetic lesion, which is absent in the control group. 4) Functional validation. The new mutations found by WES in the discovery panel and confirmed in the extension panel will be thoroughly tested in suitable in vitro, ex vivo and/or in vivo models to ascertain their driving oncogenic potential. In this regard, one of the Group Leader s (GI) Institution hosts an automated platform (available to all other groups) for testing shrna, sirna and mouse and human cdna libraries (Functional Validation laboratory, CeRMS/MBC) (Piva et al, 2006, 2010) using either cell lines or primary tumor-grafts in vitro and/or in vivo. Cell growth, viability and response to selected drugs will be determined using 96/384 plates and a multimode reader platform (SprectraMax M5/M5) and High-Content Microscope (Molecular Devices) or in vivo Imaging (MRI, PET). Functional validation studies will be also executed using a multiplexed HTP bead-based phosphomapping. The functionally validated mutations will then be exploited clinically by developing: i) in the short-term (i.e. the current grant duration), molecular and/or immunohistochemical assays for improving routine diagnosis and prognostication; and ii) in the long-term, faithful animal models and new targeted therapies. 5) Systems biology-data integration. All the available clinical information, as well as available data from other molecular and cellular analysis, will be gathered for each patient. Software already available and designed ad hoc will be used for data handling and to develop predicting models of the prognostic, diagnostic and/or therapeutic value of the candidate genes identified in the discovery/extension panels. The role of each genetic mutation in determining the diagnosis, prognosis and therapy responsiveness will be investigated by multivariate statistical analyses. Technological platforms Whole exome capture will be performed using the NimbleGen microarray (Sequence Capture 2.1M Human Exome Array) and/or the Agilent solution-based hybridization (SureSelect Human All Exon Kit) platforms, which contain probes for all coding exons and for hundreds of mirna genes. Massively parallel sequencing will then be performed using the Roche-454 (Genome Sequencer FLX Instrument/Titanium Series) or the Illumina (Genome Analyzer IIx) platforms. Moreover, we are open to use newer, more efficient and less expensive capturing and/or sequencing technologies that may become available in this rapidly evolving field during the grant s duration. The two platforms mentioned above, which offer complementary advantages in terms of read length, coverage and error type, are both already accessible to all partners of our cooperative group together with a full bio-informatics support: NimbleGen/Roche-454 is directly available at one of the Group Leaders institution (FB, ISS) and Agilent/Illumina has been successfully outsourced to a topquality service provider by another Group Leader s institution (BF, Institute of Hematology, Perugia). A dedicated unit (at the ISS) comprising bioinformaticians and biostatisticians with previous experience in data mining will be completely committed to the project. To gain deeper knowledge on the role of the mutations identified by WES, gene expression profiling analyses will be performed using the Affymetrix platform. Platforms are allocated and fully operative at different sites within the Consortium (RF, Rome; SP, Bologna; GI, Turin). Dedicated personnel is available. Codice Riferimento: 10007 Page 67 of 154
Feasibility of the project The outstanding goals of this coordinated project can only be achieved through a multi-institutional framework. All required key expertise and facilities are present throughout the Consortium and will represent the common platforms available to all groups to address the hypotheses driving this proposal: tissue and cell banking (the largest collection of archival material in Italy, as well as of primary prospective samples) and hematopathology (SP, BF, GI, GG, RF), next-generation sequencing and bioinformatics (FB), mouse modeling (GI), coordination of phase I-II clinical trials (RF). A unique asset of this multi-institutional proposal that strongly supports its transferability to the clinical setting - within the duration of the project - is the prominent power in sample collection and patients availability afforded by the center in Rome, the largest in Italy, and by the cooperative groups GIMEMA, IIL and EWALL (a Working Group of EHA stemmed from the European Leukemia Network). The PI and other Group Leaders (BF, SP, GG) are prominent coordinators/members within these networks. Over many years, GIMEMA and IIL have coordinated clinical protocols for acute and chronic leukemias and lymphomas in Italy (and Europe), and have also set up a framework for a central handling of samples from patients enrolled in the different trials. For many years, the different Group Leaders have actively collaborated. This is documented by the 169 papers in common (source PubMed). All Group Leaders are in charge of the clinical, diagnostic, prognostic, genetic and genomic facilities for hematologic malignancies within each given center. The feasibility of the project is guaranteed by the long-lasting expertise and interaction of all groups, as well as by the results already generated by the Consortium: e.g. use of TKIs in Ph+ ALL, preclinical use of TKIs, animal models, design of phase I-IV clinical trials, genetic and gene expression profile analyses, preliminary WES data, broad sample availability, etc. All the necessary facilities, competences and interactions necessary for the realization of the project are in function, including biostatisticians and bioinformaticians. Three world-known expert pathologists are Group Leaders (BF, GI, SP). Facilities include: all necessary clinical requirements - in and outpatient facilities, radiologic resources, all required laboratories and techniques (morphology, flow cytometry, cytogenetics, molecular biology, cell signaling, gene expression profiling, proteomics, MRD monitoring by flow and quantitative PCR, etc) -, banking of cell and tissue samples, WES platform, animal facilities and expertise, data center, etc. Further national and international collaborations are already ongoing (as indicated in the attached letters of collaboration). A state of the art diagnostic work-up of the different disease entities is guaranteed by the long-lasting expertise of the Group Leaders and by the network of advanced laboratories. Overall, the clinical/therapeutic component of the research program is a natural extension of the design and realization of experimental protocols that the Hematology centers in Rome and Bologna have pursued for years. The two centers have had a primary role in the development of the first targeted therapy strategies in hematology worldwide, first for APL in both adults and children (Avvisati et al, 1996; Testi et al, 2005), then in CML (Baccarani et al, 2004, 2009; Rosti et al, 2009) and, more recently, in Ph+ ALL (Vignetti et al, 2007; Foa et al, 2008). All necessary facilities - including trial office, data managers, SOP, quality controls, certified laboratories, data center, biobanking, etc are available and fully operational. Patients accrual for each disease, and subgroup entity within a given disease, is guaranteed by the flow of patients (and samples) that refer daily to the different centers and networks. The center in Rome has also a pediatric division for the supply of childhood samples. Finally, the ISS group will also ensure the necessary expertise on the regulatory issues relevant for the preparation of the dossiers for the phase I clinical studies based on the use of the new drugs designed on the basis of the driving mutations and molecular markers identified during the project. Support to the project by the participating Institutions Codice Riferimento: 10007 Page 68 of 154
The overall participating group will be composed of 78 paid collaborators, representing 85% of the total personnel dedicated to the project. The committed resources are represented by the ongoing network of dedicated laboratories, animal facilities, biobanking facilities, all necessary clinical facilities, biological and clinical data collection and analysis, WES platform, etc. Potential caveats and pitfalls Restricting the analysis to the protein coding regions (the exome ), that correspond to 1% of the complete genome is more cost effective than sequencing the whole genome and, although it has the disadvantage of excluding the structural and most noncoding variants, it is particularly valuable in studies focused on identifying somatic mutations with medical relevance, for which sample size and the interpretability of functional impact may be critical to achieve meaningful success (Albert et al, 2007). In fact, the WES mapping of genetic lesions with a filtering methodology has been shown to be a productive approach to identify candidate genes connected to human disorders even using small numbers of affected individuals (Ng et al, 2010). Nevertheless, WES will allow to identify a relatively large number of mutations. Potential caveats are represented by the fact that WES generally provides a very high amount of information whose biological meaning is not always guaranteed. Data management includes advanced and not yet harmonized competences for which excellence in information technology is required. In fact, a dedicated cross-departmental bioinformatic group with previous experience in data mining will be completely dedicated to the project. Another potential caveat is represented by the fact that, among the mutations identified, few might be recurrent among patients: to overcome this issue, once a mutation is identified in a given pathway (for example, NFkB pathway) the whole pathway will then be screened, in order to increase the possibility of identifying mutations affecting it. This approach, although time- and costconsuming, should also allow to define whether a specific compound (for example, proteasome inhibitors) might be effective in the management of the disease. Partnership The overall organization of the team is illustrated in Fig 1. The interactions will be constant, on a weekly basis. A detail work plan will be prepared. Physical meetings will be scheduled at least every three months (more frequently if required). Ad hoc meetings will be planned prior to the AIRC-defined deadlines. The PI of the project will coordinate the various activities and make sure that the advancements take place and that the milestones are realized within the expected time framework. In case of possible conflicts between partners on either the exploitation of the results or the decision on further research steps, the PI will plan a special meeting of all the partners in order to positively address these issues and take decisions; if necessary, he will ask for the advice of an external Scientific Board. In order to further facilitate the interactions between the different groups, a dedicated Intranet platform will be set up. This will enable constant contacts, exchange of information, discussion of advancements and problems, costs, reports, publication plans, etc. Intranet guarantees total privacy, otherwise not possible. In addition, the website of the Hematology Center at the Sapienza University (http://www.ematologialasapienza.org/), set up by the PI of the present project, will be utilized to release to the public domain the advancements of the work, results obtained, publications, presentations at international meetings, developments, etc. For the specific coordination of the various activities, interactions, meetings, etc of the Consortium, the PI requires a dedicated funding (specified under Meetings and travel costs ). Significance and impact Based on the results obtained in APL and CML - in terms of overall survival, chemotherapy saving and reduction of allogeneic stem cell transplant procedures - a similar scenario can be foreseen also Codice Riferimento: 10007 Page 69 of 154
for lymphoid malignancies. The preliminary results pioneered by the PI group in BCR/ABL+ ALL (the most lethal hematological malignancy) indicate that patients can be put into complete hematological remission with TKIs, without chemotherapy and partly through home care facilities (available within the Consortium). The possibility of controlling the disease without chemotherapy would have a major impact in terms of toxicity, management, applicability to the elderly (where BCR/ABL+ ALL is predominant), overall compliance, as well as outcome. The use of TK targeting in other poor prognostic patients subgroups (PTCL) opens innovative management possibilities. All the above are potential live- and toxicity-sparing procedures that can be extended worldwide and at all age ranges. This is particularly relevant, since the median life expectations, as well as the biologic age, are progressively increasing. In addition, within the 5-year extension of the project the discovery of new/driving mutations in lymphoid neoplasms, and in well defined clinical settings, will translate into new classification and prognostication schemes, more personalized treatment strategies, as well as into new specific molecular targets. Finally, the Consortium and related framework will represent an optimal setting for the enrolment of physician scientists specifically dedicated to the project. The strong, dynamic and integrated bench to bedside and vice versa structure of the project will permit a modern training of this new generation of younger clinicians, at a time when the translational nature of hemato-oncology is an absolute reality and priority. REFERENCES ALL Burmeister T, et al. Patients' age and BCR-ABL frequency in adult B-precursor ALL: a retrospective analysis from the GMALL study group. Blood,112:918,2008. Druker BJ, et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med,344:1038,2001. Foa R, et al. Dasatinib monotherapy for the 1 st line treatment of adult Ph+ acute lymphoblastic leukemia (ALL) patients: Final results of the GIMEMA LAL1205 study. Blood,112:305, 2008. Gleissner B, et al. Leading prognostic relevance of the BCR-ABL translocation in adult acute B- lineage lymphoblastic leukemia: a prospective study of the German Multicenter Trial Group and confirmed polymerase chain reaction analysis. Blood,99:1536,2002. Kurzrock R, et al. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Ann Int Med,138:819,2003. Mancini M, et al. A comprehensive genetic classification of adult acute lymphoblastic leukemia (ALL): analysis of the GIMEMA 0496 protocol. Blood,105:3434,2005. Martinelli G, et al. IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol, 27:5202,2009. Moore AS, et al. Aurora kinase inhibitors: novel small molecules with promising activity in acute myeloid and Philadelphia-positive leukemias. Leukemia,E-pub ahead of print,2010. Moorman AV, et al. Karyotype is an independent prognostic factor in adult acute lymphoblastic leukemia (ALL): analysis of cytogenetic data from patients treated on the Medical Research Council (MRC) UKALLXII/Eastern Cooperative Oncology Group (ECOG) 2993 trial. Blood,109;3189,2007. Mullighan CG, et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature,453:110,2008. Mullighan CG,et al. Deletion of IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med,360:470,2009. Codice Riferimento: 10007 Page 70 of 154
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Han X, et al. Lymphoma survival patterns by WHO subtype in the United States, 1973-2003. Cancer Causes Control,19:841,2008. Küppers R. The biology of Hodgkin's lymphoma. Nat Rev Cancer,1:15,2009. Liso A et al. Aberrant somatic hypermutation in tumor cells of nodular-lymphocyte-predominant and classic Hodgkin lymphoma. Blood,108:1013,2006. Ng AK, et al. Section VI Late Effects. In: Hodgkin Lymphoma (2 nd edition), chapters 23-27. Eds: Hoppe RT, et al. Lippincott Williams&Wilkins, 2007. Swerdlow SH, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (4 th edition). IARC press, 2008. FEASIBILITY OF THE PROJECT Avvisati G, et al. AIDA (all-trans retinoic acid + idarubicin) in newly diagnosed acute promyelocytic leukemia: a Gruppo Italiano Malattie Ematologiche Maligne dell'adulto (GIMEMA) pilot study. Blood,88;1390,1996. Baccarani M, et al. GIMEMA Working Party on Chronic Myeloid Leukemia. Imatinib and pegylated human recombinant interferon-alpha2b in early chronic-phase chronic myeloid leukemia. Blood,104;4245,2004. Baccarani M, et al. Comparison of imatinib 400 mg and 800 mg daily in the front-line treatment of high-risk, Philadelphia-positive chronic myeloid leukemia: a European LeukemiaNet Study. Blood,113;4497,2009. Rosti G, et al. GIMEMA CML Working Party. Nilotinib for the frontline treatment of Ph(+) chronic myeloid leukemia. Blood,114;4933,2009. Testi AM, et al. GIMEMA-AIEOPAIDA protocol for the treatment of newly diagnosed acute promyelocytic leukemia (APL) in children. Blood,106;447,2005. POTENTIAL CAVEATS AND PITFALLS Albert TJ, et al. Direct selection of human genomic loci by microarray hybridization. Nat Methods,4:903-5,2007. Ng SB, et al. Exome sequencing identifies the cause of a mendelian disorder. Nat Genet,42:30-5,2010. Codice Riferimento: 10007 Page 73 of 154