Genetics-driven targeted management of lymphoid malignancies

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1 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: Page 48 of 154

2 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 (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 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: Page 49 of 154

3 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: Page 50 of 154

4 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: Page 51 of 154

5 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: Martinelli G et al. J Clin Oncol 2009;27: Rossi D et al. Clin Cancer Res 2009;15: Kuppers R. Nat Rev Cancer 2009;9: Ley TJ et al. Nature. 2008;456: Swerdlow SH et al. WHO Press, 2008; pp Vignetti M et al. Blood 2007;109: Piccaluga PP et al. J Clin Invest 2007;117: Piva R et al. J Clin Invest 2006;116: Codice Riferimento: Page 52 of 154

6 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: Page 53 of 154

7 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: Page 54 of 154

8 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: Page 55 of 154

9 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: Page 56 of 154

10 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 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: Page 57 of 154

11 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: Page 58 of 154

12 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: Page 59 of 154

13 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: Page 60 of 154

14 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 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: Page 61 of 154

15 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: Page 62 of 154

16 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: Page 63 of 154