PROJECT CATEGORIES

ERICLL DATABASE

TARGETED RE-SEQUENCING IN CLL

Comparative analysis of target enrichment techniques for the detection of gene mutations in CLL

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)

Lesley Ann Sutton; Richard Rosenquist; Affiliations: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

Background

Targeted NGS, in which a select number of genes are sequenced, is emerging as a much more efficient means to identify genetic variants and contains many attractive features, including: 1) the capacity to custom design a gene panel and screen a large number of genes and samples simultaneously; 2) the ability to detect mutations present at low variant allelic frequencies due to the high sequence depth achievable; and 3) the possibility to analyse all coding exons within a gene, irrespective of its length. As a consequence, this methodology is now being considered within everyday clinical practice. However, with several enrichment techniques now available for targeted sequencing, prior to implementing such a workflow, questions that commonly arise concern the specific technique that should be chosen, whether differences in sensitivity/specificity exist between the various techniques, which genes should be analysed, the optimal number of samples to multiplex, the appropriate bioinformatics workflow etc.

Main objectives

Motivated by this unmet need and uncertainty, ERIC has launched an initiative aimed at simplifying the introduction of targeted NGS into clinical laboratories by identifying the pros or cons (if any) of various enrichment techniques and determining the most suitable for the detection of recurrent mutations in CLL as well as ensuring end-to-end harmonization in the diagnostic approach.

Main activities

Three enrichment techniques (Haloplex, Agilent; TruSeq, illumina; CLL MASTR plus, Multiplicom) will be compared. Each technique will be analyzed by at least 2 centers and all sequencing will be performed on the Illumina MiSeq instrument. Eleven genes recurrently mutated in CLL will be included in the analysis. Forty-eight CLL samples with matched germline DNA will be included in the study (all centers will analyse the same samples). To be included in the study a sample must be well characterized and carry a mutation in at least one of the selected genes.

Project Start date           1 May 2015

Project Finish date         30 November 2017

Different phases within the project

Phase 1 – comparative analysis of target enrichment techniques;

Phase 2 – large-scale validation of a gene panel for CLL diagnostics;

Phase 3 – longitudinal studies using NGS technologies

Contact person (s) for further details

Lesley Ann Sutton; lesley.sutton@igp.uu.se

Recurrent gene mutations in CLL: ERIC project in HARMONY

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)

Lesley Ann Sutton; Richard Rosenquist; Affiliations: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

Background

Next-generation sequencing (NGS) technologies have proved invaluable for deciphering the molecular heterogeneity of chronic lymphocytic leukemia (CLL). Such studies have revealed a diverse pattern of mutations, with a limited number of frequently mutated genes identified followed by a seemingly endless tail of genes harboring mutations at an extremely low frequency. Evidence suggests that this observed biological heterogeneity is intricately linked to the heterogeneous pathological characteristics and clinical course evidenced in CLL patients. Although certain gene mutations have prognostic or even predictive capabilities, most gene mutations require additional rigorous validation in large, patient series, to not only discern their potential contribution (if any) to the pathobiology of CLL but also ascertain whether a particular gene mutation could aid in clinical decision-making, including therapy selection and response prediction.

Main objectives             

We will evaluate the mutational status of genes in a well-characterized cohort encompassing thousands (≈4000) of CLL patients through a coordinated, multi-center collaboration conducted under the auspices of the European Research Initiative in CLL (ERIC). More specifically, we will investigate: (i) the most recurrently mutated genes in CLL, namely NOTCH1, SF3B1, ATM, BIRC3, MYD88 and TP53; mutations within the TP53 and ATM genes have long been known to confer poor prognosis, however recurrent mutations within NOTCH1, SF3B1 and BIRC3 have more recently been reported to be associated with poor clinical outcome with higher frequencies in relapsing/refractory CLL and in Richter’s syndrome; and, (ii) an additional four genes, i.e. EGR2, NFKBIE, POT1 and XPO1; recurrent mutations in these genes have been associated with a dismal clinical course, although they have been less studied.

Main activities

  1. Evaluation of the mutational status of NOTCH1, SF3B1, ATM, BIRC3, MYD88, TP53, EGR2, NFKBIE, POT1 and XPO1 in a large series of CLL cases, well-annotated for both molecular parameters and clinical characteristics.
  2. Assessment of the prognostic impact and clinical relevance of recurrent gene mutations.
  3. Identification of distinct patterns of associations between recurrent mutations with other clinicobiological features in CLL.
  4. Perform robust validation of recently proposed prognostication models that incorporate both cytogenetic and molecular lesions as well as immunogenetic characteristics.

Project Start date           1 January 2017

Project Finish date         31 May 2018

Name of sponsor (s) if any

Forms the pilot CLL project within the Harmony alliance; a European Network of Excellence in Hematology and Big Data that is funded through the Innovative Medicines Initiative (IMI)

Contact person (s) for further details

Lesley Ann Sutton; lesley.sutton@igp.uu.se

Prevalence of BTK and PLCϒ2 Mutations in Patients relapsing under Ibrutinib

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)

Paolo Ghia; IRCCS San Raffaele Scientific Institute, Milan, Italy.

Background

The first-in-class irreversible BTK inhibitor, ibrutinib, showed impressive efficacy in CLL leading to FDA and EMA approval. Late relapses on ibrutinib often associate with mutations within the Bruton’s tyrosine kinase (BTK) gene at the binding site of ibrutinib or within the PLCγ2 gene, that is located immediately downstream of BTK. Deep sequencing analysis confirmed that up to 80-85% of resistant cases harbor mutations within BTK and/or PLCG2 in a variable proportion of CLL cells in resistant cases, ranging from 0.1 to 80% [according to the estimated variant allele frequency (VAF)]. As CLL patients relapsing on ibrutinib represent a new unmet clinical need, thorough understanding of the mechanisms behind the development of resistance as well as the actual prevalence of the mechanisms so far identified is key to assist treatment decisions and improve the outcomes of such patients. To this aim, we propose this retrospective case-based study, aimed at characterizing the prevalence of BTK and PLCG2 mutations in CLL patients relapsing on ibrutinib and at investigating additional potential mechanisms of resistance.

Main objectives             

Primary Objective: To define the prevalence of BTK and/or PLCG2 mutations in chronic lymphocytic leukemia (CLL) patients relapsing under ibrutinib.

Secondary Objectives: To define the prevalence of BTK and/or PLCG2 mutations in CLL patients responding to ibrutinib after at least 1 year of treatment; To define the estimated variant allele frequency (VAF) in CLL patients relapsing under ibrutinib and in those responding to ibrutinib after at least 1 year of treatment.

Main activities

Targeted next-generation sequencing (NGS) of the BTK and PLCG2 genes.

Exploration of statistical associations with various clinicobiological features, including: demographic data; disease characteristics; previous treatment and ibrutinib treatment information.

Project Start date           November 2017

Project Finish date        June 2018

Different phases within the project     

Sample collection will start in November 2017. Library preparation and Sequencing will start in December 2017. Data analysis will start February 2018. Completion of the project is expected by June 2018.

Contact person (s) for further details  

Lydia Scarfò; scarfo.lydia@hsr.it; Silvia Bonfiglio; bonfiglio.silvia@hsr.it


IMMUNOGLOBULIN GENE SEQUENCES

BcR IG sequence collection

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)     

Kostas Stamatopoulos; Andreas Agathangelidis; Affiliation: Centre for Research and Technology Hellas (CERTH) / Institute Of Applied Biosciences (INAB),  Thessaloniki , Greece

Background      

Immunogenetic analysis of the B cell receptor immunoglobulin (BcR IG) has been instrumental for understanding the pathogenesis of chronic lymphocytic leukemia (CLL) and has also helped in the classification of patients into groups with distinct prognosis. A highly restricted BcR IG gene repertoire was the first evidence for antigen selection in the natural history of CLL. Additional evidence was provided by the differential prognosis of cases with distinct somatic hypermutation load within the clonotypic immunoglobulin heavy variable (IGHV) genes. Yet, perhaps the strongest evidence for the importance of microenvironmental interactions in driving CLL development and evolution is the existence of subsets of patients with stereotyped BCRs, collectively accounting for a remarkable one-third of the entire cohort. These observations have been instrumental in shaping the notion that CLL ontogeny is functionally driven and dynamic, rather than a simple stochastic process.

Main objectives             

In collaboration with IMGT®, the International Immunogenetics Information System, this ERIC project entails the expansion and maintenance of the IMGT/CLL-DB (http://www.imgt.org/CLLDBInterface/query), a database holding primary IG sequences from patients with CLL to foster high-quality collaborative research on CLL and related disorders, particularly regarding the pathogenesis and molecular epidemiology of CLL.

Main activities

Main activities include: (i) data acquisition, (ii) data processing and curation, (iii) data upload on IMGT/CLL-DB, (iv) data analysis.

Project Start date           1 January 2018

Project Finish date         31 December 2025 

Different phases within the project     

Phase 1: data collection,

Phase 2: data curation,

Phase 3: upload to the IMGT/CLL-DB,

Phase 4: BcR IG repertoire analysis.

Name of sponsor (s) if any        

Janssen

Contact person (s) for further details  

Andreas Agathangelidis; aagathangelidis@gmail.com

FLOW CYTOMETRY FOR DIAGNOSIS AND MONITORING

CLINICAL PROJECTS

International CLL registry for spontaneous regression in CLL

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)

Ilaria Del Giudice, Emili Montserrat IDG: Affiliations; Division of Hematology, Department of Cellular Biotechnologies and Hematology, “Sapienza” University, Rome (Italy); Hospital Clinic Barcelona, Barcelona (Spain)

Background

Spontaneous regression in CLL in the absence of any previous treatment is exceptional and the biologic bases are unknown (Del Giudice et al. Blood 2009). Since the incidence of CLL spontaneous regression has been estimated about 1%, no single center will have many patients with this infrequent event. The proposal of an international registry for spontaneous regression in CLL has been launched among the ERIC members. The aim is to pool clinical data and biological information of these cases and making them accessible to many researchers for future studies.

Main objectives             

  • identify key differences with CLL cases which follow the usual natural history of the disease
  • formulate hypothesis on the pathogenesis of this phenomenon and on mechanism of control of CLL progression to more advanced stages.

Main activities

A dedicated form to collect clinical data (medical history, drug intake, biologic markers, etc.) is available in the website. Due to the rarity of this condition, the project is still open and running. So far, 24 new cases with spontaneous regression have been included in the registry, 15 from Spain, 7 from Italy and 2 from Sweden. A new call has been launched in these days to researchers interested in CLL.

In the case peripheral blood samples can be obtained, biological studies on the residual CLL clone and on the T cell populations will be performed.

Project Start date           15 February 2011

Project Finish date         15 December 2018

Different phases within the project

Phase I (collection of clinical data in the registry). Likely, the registry will be closed by the end of 2018.

Phase II (collection of peripheral blood samples for biologic studies) will be eventually opened afterwards.

Contact person (s) for further details

Ilaria Del Giudice, delgiudice@bce.uniroma1.it   

Observational study to assess the efficacy and safety of bendamustine plus rituximab in patients affected by chronic lymphocytic leukemia

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)     

Antonio Cuneo; Affilliation: University of Ferrara, Ferrara (Italy)

Background      

The efficacy and safety of the BR regimen in first line and in second line in the clinical practice is not defined

Main objectives             

The primary objective of the study is to observe the progression-free survival in CLL patients treated with first and second-line BR regimen.

Main activities  compilation of the CRF

Project Start date           10 January 2016

Project Finish date         Q1 2018

Different phases within the project     

1) Collection of the data

2) Writing of the manuscript

Name of sponsor (s) if any        

GIMEMA-ERIC

Contact person (s) for further details  

Antonio Cuneo; cut@unife.it

CLL Patient Empowerment Program

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)     

Aliki Xochelli, Christina Karamanidou, Kostas Stamatopoulos (Affiliation: Centre for Research and Technology Hellas (CERTH) / Institute Of Applied Biosciences (INAB), Thessaloniki, (Greece)

Background      

A paradigm shift described in healthcare indicates that preventive, pre-emptive and predictive healthcare decisions should be made in a pervasive, participatory and personalized manner. It is widely accepted that such a paradigm shift is not possible using traditional tools and processes and requires the patient’s active engagement in the management of his/her own health. Evidently, patient empowerment towards self-management requires novel methodological and technological frameworks, well-designed educational programs that will familiarize patients, caregivers and healthcare professionals alike with the concept of co-decision, its scope and challenges but also its misuse. We herein envision patient empowerment as a four-arm, iterative process that involves patients, caregivers and healthcare professionals, namely physicians and nurses, and propose a framework for chronic lymphocytic leukemia (CLL), the most frequent adult hematologic cancer. Our CLL patient empowerment program aims to engage physicians and nurses, empower and support patients and caregivers and promote co-decisions between physicians and patients by establishing a common language and structured ways of communication.

Main objectives             

  1. To explore CLL patients’ experience of illness (physical, emotional, social dimension), and specifically their understanding of the disease (physical dimension), their ways of coping, perceived impact etc. Also, their experience regarding treatment, their understanding and concerns as well as their relationship with physicians etc.
  2. To explore health care professionals experience in treating patients with CLL and identify their needs and concerns with regards to communication within the context of patient-physician relationship.
  3. To systematically record patients’ beliefs, knowledge and preferences regarding their illness and treatment, their mood, their satisfaction concerning medical information, their adherence to medication etc.
  4. To systematically record patient reported outcomes regarding symptoms, adverse effects and life style changes throughout the disease course.
  5. To develop an educational framework for (i) the enhancement of physicians communication skills and (ii) the enhancement of patients self assessment and self management and also their caregivers knowledge on CLL

Main activities               

  • Face to face interviews/ focus groups with participants (health care professionals and patients/caregivers)
  • Self-completed Questionnaires −
  • Development of a CLL knowledge questionnaire and a treatment preference questionnaire for patients –
  • Design, development and evaluation of tools that will support physician communication and patient empowerment

Project Start date           1 January 2017

Project Finish date         1 January 2020

Different phases within the project     

Phase I- Needs assessment phase

Phase II- Design and development of tools

Phase III- Evaluation of tools

Name of sponsor (s) if any        

Janssen Global ; Gilead Sciences Hellas; Janssen Cilag Greece ;

Contact person (s) for further details 

Aliki Xochelli; aliki.xochelli@gmail.com; Christina Karamanidou; ckaramanidou@gmail.com

CYTOGENETIC COMPLEXITY IN CLL

Prognostic and predictive value of complex karyotyping using chromosome banding analysis or microarray-based profiling in CLL

Project Leader (s), Coordinator (s) or/and Principal Investigator (s)

Arnon Kater; Kostas Stamatopoulos; Affiliations: Academic Medical Center Amsterdam – University of Amsterdam, Amsterdam, (The Netherlands); Centre for Research and Technology Hellas (CERTH) / Institute Of Applied Biosciences (INAB), Thessaloniki, (Greece)

Background

Today, the ‘gold standard’ for genetic risk stratification in CLL is FISH analysis with probes for chromosomes 11q, 12, 13q and 17p complemented by mutational screening for the TP53 gene found to be recurrently mutated in CLL. Such screening (particularly for del17p and TP53 mutations) has helped to select patients with low versus high response chances to standard chemo-immunotherapy. This is especially relevant considering the rapid expansion of effective but highly expensive alternative treatment regimens in high-risk patients. Therefore, improved risk-stratification has become a priority in CLL patient management.

Studies from the early 1990s provided the first evidence for the turbulent genetic landscape of CLL as revealed by classic chromosome banding analysis (CBA). Against this evidence, FISH superseded CBA in the routine diagnostic setting as the latter was more laborious, considering the need of in vitro stimulation in order to obtain mitoses. That notwithstanding, FISH is assessing only selected chromosomal regions whereas CBA offers the opportunity to globally assess the karyotype of the malignant clone. Indeed, by providing a more comprehensive view of the genetic landscape, CBA may offer valuable complementary information to the routinely performed FISH analysis in CLL, potentially, improving risk stratification. This is especially so today, when effective and reliable cell stimulation protocols are available, overcoming the limitations of older protocols.

More recently, CBA in CLL has attracted great interest in view of reports indicating: complex karyotype (CK) as a potential predictive marker for refractoriness to ibrutinib, independently of the presence of the chromosome 17p deletion [del(17p)]; and, lack of any adverse prognostic effect of CK on either PFS or OS, irrespective of the presence of del(17p)/TP53mut, in idelalisib-treated patients. That said, the available evidence does not yet allow reaching definitive conclusions regarding either the precise role of signaling inhibitors at large in the management of CLL patients with CK or the relative value of one inhibitor over the other in this particular group of patients.

Altogether, these findings allude to the important potential role of comprehensive karyotype analysis for refined risk stratification in CLL. However, many challenges towards routine clinical application of CBA still need to be overcome, thus indicating the urgent need for rigorous definitions, particularly regarding which cytogenetic profiles exactly correspond to CK, validated methodologies and systematic investigation in large series.

Array-based analysis is another powerful technique capable of globally assessing the CLL genome. From a technical perspective, in contrast to CBA, no cell stimulation step is needed for array-based analysis which also offers increased detection depth regarding deletions or duplications. However, balanced aberrations cannot be detected by micro-arrays, which is not trivial, especially considering reports highlighting their potential prognostic impact on clinical outcome.

The application of array-based analysis in CLL not only revealed the well-known recurrent chromosome abnormalities but also recovered additional (recurrent) genetic alterations, including complex genomic profiles with 3 or more acquired copy number aberrations (aCNAs). Elevated CLL genomic complexity demonstrated by microarrays identified patients with aggressive CLL and short survival.

Main objectives             

  1. To compare with regards to the detection rate of CK:
  • different CBA stimulation protocols [CpG-oligodeoxynucleotides/interleukin 2 (CpG/IL2), phorbol-12-myristate-13-acetate (TPA) or both]
  • CBA versus microarray analysis in well annotated patient samples.
  1. To assess the prognostic and predictive relevance of various numerical cut-offs for cytogenetic aberrations (3 vs 4 vs 5 or more) for defining cytogenetic complexity and investigate the importance of the type of aberrations (i.e. structural versus numerical).
  2. To assess the potential associations between CK and other genetic features of the malignant clones, particularly the presence of TP53 aberrations (deletion of chromosome 17p and/or TP53 mutation).
  3. To determine the precise prognostic role of CK in risk stratification of patients with CLL.

Main Activities

  • Gather and analyze CBA data as performed in labs through Europe
  • Gather and analyze array data as performed in labs through Europe
  • Define exact definitions of CK in CLL
  • Compare the reproducibility of the 2 techniques

Project Start date           1 February 2016

Project Finish date         1 December 2018

Different phases within the project

  • Analysis of CBA data from different labs in Europe
  • Analysis of array data from different labs in Europe
  • Harmonization activities
  • CBA and array analysis of CLL patients treated with novel biological agents

Name of sponsor (s) if any

Janssen

Contact person (s) for further details

Arnon Kater; a.p.kater@amc.nl; Kostas Stamatopoulos; kostas.stamatopoulos@certh.gr