CLONAL HETEROGENEITY IN LYMPHOID NEOPLASMS
UNIVERSIDAD AUTONOMA DE MADRID
FACULTAD DE CIENCIAS
DEPARTAMENTO DE BIOLOGÍA MOLECULAR
JULIA GONZÁLEZ RINCÓN
Madrid 2018
CLONAL HETEROGENEITY IN LYMPHOID NEOPLASMS
UNIVERSIDAD AUTONOMA DE MADRID
FACULTAD DE CIENCIAS
DEPARTAMENTO DE BIOLOGÍA MOLECULAR
JULIA GONZÁLEZ RINCÓN
Licenciada en Biotecnología Madrid 2018
DIRECTORA DE TESIS
Dra. Margarita Sánchez-Beato Gómez
This thesis, submitted for the degree of Doctor of Philosophy at the Universidad Autónoma de Madrid, has been performed in the Lymphomas Laboratory at Instituto de Investigatición Sanitaria Puerta de Hierro-Segovia de Arana. This work was supported by the Spanish Ministry of Economy and Competence, ISCIII-AES-FEDER (Plan Estatal de I+D+I 2008-2011 and 2013- 2016) (RD12/0036/0060, PI10/00621 PI14/00221, CIBERONC CB16/12/00291, DTS17/00039) JGR has been supported by an iPFIS predoctoral fellowship (ISCIII-MINECO AES-FEDER, IFI14/00003) and Fundación de Investigación Biomédica HU Puerta de Hierro-Majadahonda.
Esta tesis, presentada para optar al grado de doctor en la Universidad Autónoma de Madrid, ha sido realizada en el laboratorio de Linfomas del Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana. La realización de este trabajo ha sido posible gracias a la financiación del Ministerio de Economía y Competencia, ISCIII-MINECO AES-FEDER (Plan Estatal de I+D+I 2008-2011 and 2013-2016: RD12/0036/0060, PI10/00621 PI14/00221, CIBERONC CB16/12/00291, DTS17/00039). JGR ha sido beneficiara de un contrato predoctoral iPFIS, financiado por ISCIII-MINECO AES-FEDER (IFI14/00003) y la Fundación de Investigación Biomédica HU Puerta de Hierro-Majadahonda.
TABLE OF CONTENTS
ABSTRACT... 5
RESUMEN ... 7
ABREVIATIONS ... 11
1. INTRODUCTION ... 15
Lymphoid Malignancies ... 15
Non-Hodgkin Lymphomas ... 16
B-cell Neoplasms ... 17
B-Cell Chronic Lymphoid Leukaemia/Small Lymphocytic Lymphoma... 18
General Features ... 18
Pathology ... 18
Prognostic Markers ... 19
Classical Staging Systems ... 19
IGHV Status ... 20
Immunophenotypic Markers ... 20
Cytogenetics ... 21
New Staging Score ... 21
Genetic Landscape ... 22
Minimal Residual Disease in CLL... 22
Follicular Lymphoma ... 23
General Features ... 23
Clinical Features ... 23
Staging score ... 24
Pathology ... 25
Genetics and Cytogenetics ... 26
Follicular Lymphoma Transformation ... 27
General Features ... 27
Genetics and Cytogenetics ... 28
Diffuse Large B Cell Lymphoma Transformed from FL ... 29
Tumour Heterogeneity and Clonal Evolution ... 30
Genomics: Next-Generation Sequencing ... 33
2. HYPOTHESES AND OBJECTIVES ... 37
Project I ... 37
Project II ... 38
3. MATERIALS AND METHODS ... 41
Patients and Cell Lines ... 41
Patients and samples - Project Ia ... 41
Patients and samples - Project Ib ... 41
Patients and samples– Project II ... 44
Cell lines and culture conditions – Project II ... 45
Clinical, Molecular and Pathological data ... 46
Clinical data ... 46
Project I ... 46
Extraction of DNA from peripheral blood mononuclear cells and cell lines ... 49
Extraction of DNA from Formalin Fixed Paraffin Embedded Tissue ... 49
Extraction of RNA from cell lines ... 49
Massive sequencing techniques and bioinformatics analysis ... 50
Target Sequencing - Project Ia ... 50
Haloplex Target Enrichment -CLL panel ... 50
Custom Sequencing of EGR2 Hotspot ... 50
Data Analysis and Variant Calling – Project Ia ... 51
Target Sequencing - Project Ib ... 52
Whole-Exome Sequencing ... 52
TruSeq Target Sequencing – CLL Validation Panel ... 53
Target Sequencing - Project II ... 54
Haloplex Target Enrichment – FL Panel for Ion Proton ... 54
SureSelect Target Enrichment – FL Panel for Illumina platforms ... 54
Sanger Sequencing ... 56
SNP arrays ... 56
Western Blot ... 57
Quantitative RT-PCR ... 57
Statistical Analysis ... 58
4. RESULTS ... 62
PROJECT Ia ... 62
Mutational Landscape of progressed CLL patients ... 62
Correlation with patients’ cytogenetic and phenotypic features ... 63
Patterns of clonality ... 64
Correlation with clinical follow-up and response to therapy ... 65
PROJECT Ib ... 69
Whole exome sequencing in index samples ... 69
Clonal heterogeneity and evolution ... 69
Patient 1 ... 69
Patient 2 ... 72
PROJECT II ... 74
Recurrent genetic alterations in transformed follicular lymphoma ... 74
Clonality and Evolution ... 76
Recurrent splicing mutations in POU2AF1... 77
Genetic lesions associated with transformation ... 78
5. DISCUSSION ... 83
PROJECT Ia ... 83
PROJECT Ib ... 85
PROJECT II ... 88
Clonal heterogeneity in progression and transformation ... 91
Selective pressure among existing alterations ... 92
DNA damage, introduction of new mutations ... 92
Pathways and cellular mechanisms altered in progression and/or transformation ... 93
6. CONCLUSIONS ... 97
7. REFERENCES ... 101
ANNEX ... 118
ABSTRACT
ABSTRACT
Tumour heterogeneity is defined as the presence of subpopulations of cells possessing different genomic alterations within a tumour and has been associated with progression, transformation and poor clinical outcome. The growth in the access to deep sequencing techniques is increasing the knowledge about the prevalence of intra and inter-tumour heterogeneity. This thesis studies the molecular mechanisms involved in progression and transformation through the study of two indolent B-cell lymphomas: chronic lymphocytic leukaemia (CLL) and follicular lymphoma (FL).
CLL is the most prevalent leukemia in Western countries. It is incurable and characterized by a highly variable clinical outcome. It is an ideal model for studying clonal heterogeneity and dynamics during cancer progression, response to therapy and/or relapse, because the disease usually develops over several years.
We have studied progression in CLL by assessing the mutational status of 26 CLL recurrently mutated genes by deep-targeted sequencing in 90 CLL samples from patients with active progressive disease prior to first line treatment. Seventy-one patients were enrolled in the REM (Rituximab in Maintenance) clinical trial. We found that the frequency of mutations in NOTCH1 (26,6%), SF3B1 (20%) and ATM (16,6%) were higher in these progressed patients in need of treatment than in other more heterogeneous series when analyzing CLL-diagnostic samples. Minimal residual disease detection at the end of treatment was more frequent in TP53- and NOTCH1-mutated cases. EGR2 mutations were identified as an independent unfavorable marker for progression-free survival.
For a better insight about the evolution through disease progression and response to treatment, we have studied two CLL patients with 12- and 7-year disease courses by deep sequencing of sequential samples taken at different times from the affected organs. One of the patients followed a linear pattern of clonal evolution, acquiring and selecting new mutations in response to salvage therapy and/or allogeneic transplantation, while the other suffered loss of cellular tumoral clones during progression and histological transformation.
FL is an indolent, but mainly incurable disease. Some patients suffer histologic transformation to a more aggressive subtype with poorer prognosis. Targeted massive parallel sequencing was conducted for 22 pre-transformed FL / transformed diffuse large B-cell lymphoma (DLBCL) pairs and 20 diagnostic samples from non-transformed FL patients. Recurrently mutated genes associated with transformation were identified, most notably LRP1B, GNA13 and POU2AF1, which have roles in B-cell differentiation, GC architecture and migration. We observe a more complex mutational landscape in pre-tFL vs. ntFL and have also identified four genes (NOTCH2, DTX1, UBE2A and HIST1E1) whose mutations in FL samples are associated to transformation. The result also pointed out a putative role for Notch pathway alterations in transformation.
RESUMEN
La heterogeneidad tumoral se define como la presencia de subpoblaciones de células en el tumor con distintas alteraciones genómicas y se ha asociado con progresión, transformación y curso clínico desfavorable. El creciente acceso a las técnicas de secuenciación masiva (SM) ha aumentado el conocimiento sobre la heterogeneidad intra- e inter-tumoral. Esta tesis estudia esta heterogeneidad y su papel en progresión y transformación tumoral mediante el estudio de dos linfomas B indolentes: la leucemia linfática crónica (LLC) y el linfoma folicular (LF).
La LLC es la leucemia más prevalente en los países occidentales. Es incurable y se caracteriza por tener un curso clínico muy variable. Es además un modelo ideal para el estudio de la heterogeneidad y dinámica clonal en la progresión del cáncer, respuesta a tratamiento y/o recaída, debido a la facilidad de acceso a la muestra tumoral y a que se desarrolla a lo largo de muchos años.
En esta tesis, se ha estudiado la progresión de la LLC analizando el estado mutacional de 26 genes utilizando SM en 90 muestras de pacientes con LLC y enfermedad progresiva activa previas al inicio de la primera línea de tratamiento. Setenta y uno de los 90 pacientes se reclutaron en el ensayo clínico REM (Rituximab en mantenimiento). La frecuencia de mutaciones en los genes NOTCH1 (26,6%), SF3B1 (20%) y ATM (16,6%) fue superior en esta serie de pacientes progresados con necesidad de tratamiento que en otras series más heterogéneas que analizaban muestras al diagnóstico. La detección de la enfermedad mínima residual al final del tratamiento fue más frecuente en los casos mutados en TP53 y NOTCH1; además las mutaciones en EGR2 son un marcador independiente desfavorable para la supervivencia libre de progresión.
Se han estudiado también dos casos de LLC con 12 y 7 años de evolución mediante SM de alta profundidad en muestras secuenciales tomadas a diferentes tiempos y de diferentes órganos afectados. La evolución clonal en uno de los pacientes sigue un patrón lineal, adquiriendo y seleccionando nuevas mutaciones en respuesta a quimioterapia de altas dosis y/o trasplante alogénico, mientras que el otro sufre pérdida de un clon tumoral y ganancia de otros durante su progresión y transformación histológica.
El LF es un linfoma indolente, mayoritariamente incurable. Algunos pacientes sufren transformación histológica a un subtipo más agresivo con peor pronóstico. Se secuenciaron muestras pareadas de 22 pacientes (de diagnóstico de LF y de la transformación) y 20 muestras de pacientes con LF no transformado. Se han identificado mutaciones en genes más frecuentes en la transformación, como LRP1B, GNA13 y POU2AF1, con funciones en la diferenciación de la célula B, arquitectura del centro germinal y migración celular. Se ha observado mayor complejidad de alteraciones genéticas en los FL
ABREVIATIONS
ABREVIATIONS
Activated B-cell like ABC
Aberrant somatic hypermutation aSHM
B allele frequency BAF
B cell lymphoma BCL
B-cell-receptor BCR
B cell non-Hodgkin lymphoma B-NHL
Burrows-Wheeler Aligner BWA
Common Gene Set CGS
Cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, prednisone CHOP
Chemo-immunotherapy CIT
Chronic lymphocytic leukemia CLL
CLL-International Prognosti Index CLL-IPI
Chromatin modifying gene CMG
Centro Nacional de Análisis Genómico CNAG
Central nervous system CNS
Copy number variations CNV
Cell of Origine COO
Catalogue of Somatic Mutations in Cancer COSMIC
Common progenitor cell CPC
Cyclophosphamide, prednisone CVP
B-2 microglobulin D2M
Diffuse large B-cell lymphoma DLBCL
Deoxyribonucleic acid DNA
Epstein-Barr virus (EBV)-encoded small RNAs EBERs
Eastern Cooperative Oncology Group ECOG
European Medicines Agency EMA
Fetal bovine serum FBS
Fludarabine, cyclophosphamide and rituximab FCR
Follicular dendritic cells FDC
Formalin Fixed Paraffin Embedded FFPE
Formalin-fixed, paraffin-embedded tissue FFPET
Fluorescence in Situ Hybridization FISH
Follicular lymphoma FL
Follicular Lymphoma International Prognostic Index FLIPI
Genome Analysis Toolkit GATK
Germinal center GC
Germinal center B-cell like GCB
Histologic transformation HT
International Cancer Genome Consortium ICGC
Immunoglobulin heavy-chain variable IGHV
Integrative Genome Viewer IGV
Linfoma folicular LF
Leucemia Linfática Crónica LLC
Lymph Node LN
Log R ratio LRR
Mucosa-associated lymphoid tissue MALT
Minimal Residual Disease MRD
Next Generation Sequencing NGS
Non-Hodgkin lymphoma NHL
Overall survival OS
Peripheral Blood Mononuclear Cells PBMCs
Polymerase chain reaction PCR
Progression Free Survival PFS
Rituximab R
Ribonucleic acid RNA
Reverse transcription polymerase chain reaction RT-PCR
Stem cell transplant SCT
Somatic hypermutation SHM
Secuenciación Masiva SM
Single Nucleotide Polymorphisim SNP
Sequence Read Archive SRA
T cell lymphoma TCL
Tranformated follicular lymphoma tFL
Time to transformation TTT
Variant Allele Frequency VAF
Whole Exome Sequencing WES
World Health Organization WHO
INTRODUCTION
1. INTRODUCTION
Lymphoid Malignancies
Lymphoid malignancies comprise one of the most heterogeneous sets of diseases classified as a single type of malignancy. These cancers arise from the malignant transformation and uncontrolled proliferation of B, T and NK cells, and they can form tumours in any organ in the body, including lymph nodes, spleen, blood, bone marrow and other organs (Swerdlow et al., 2016).
Insights into the normal immune system have improved our understanding of these sometimes confusing disorders. In fact, lymphomas are stratified according to the origin of their cell lineage and whether they are derived from immature or mature lymphocytes.
Classification criteria are based on morphology, immunophenotype, genetic abnormalities and molecular and clinical features (“The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. - PubMed - NCBI,” 2018, “WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues” 2018). They are traditionally divided into Hodgkin lymphoma (HL), which accounts for about 10% of all lymphomas and typically have a good prognosis, and non-Hodgkin lymphoma (NHL), which is subclassified by lymphoma cell type (B, T and NK) and into indolent and aggressive types (Swerdlow et al., 2016).
Malignant lymphomas are associated with recurrent genetic abnormalities. These abnormalities can be identified at a variety of levels, including those of gross chromosomal changes (translocations, amplifications and deletions); overexpression, underexpression, or mutation of specific oncogenes. Translocations, altered expression and mutation of specific proteins are particularly important.
Non-Hodgkin Lymphomas
About 85-90% of NHLs are derived from B cells, whereas the other lymphomas are derived from T cells or NK cells (Armitage et al., 2017; “WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues” 2018). NHL encompasses a spectrum of cancers of the immune system that vary from the most indolent to the most aggressive malignancies (Figure 1.1). They arise from lymphocytes that are at various stages of development, and the characteristics of the specific lymphoma subtype reflect those of the cell from which they originated.
Figure 1.1 Classification of non-Hodgkin lymphomas (neoplasms studied in this thesis in bold).
The human immune system has the remarkable ability to respond in a specific and adaptive way to an essentially infinite number of possible antigens. This diversity of specificity is achieved by establishing a repertoire of B-cell clones, each expressing a unique B-cell receptor (BCR). Between them, they have the ability to bind to all possible antigens whilst the processes that rearrange the immunoglobulin genes are required to generate BCR diversity. It is the breakdown, dysregulation and off-target effects of these processes that give rise to the genetic lesions seen in B-cell non-Hodgkin lymphomas (B-NHLs) (Blombery et al., 2015).
Immunoglobulin gene rearrangements arise during B lymphoid development. This process may contribute to immunoglobulin heavy-chain locus chromosomal translocations in
immune responses, and may explain the link between immunity and lymphoid neoplasia (Macintyre et al., 2000).
The majority of NHLs undergo clonal immunoglobulin (Ig) or T cell receptor (TCR) rearrangements. The identification of a clonal Ig/TCR rearrangement (lymphoid clonality) is used at diagnosis and for follow-up. It is probably the most frequently performed diagnostic molecular analysis with NHL (Jung and Alt, 2004).
B-cell Neoplasms
B-NHLs are a diverse group of haematological malignancies that are characterised by a broad range of morphological, immunophenotypic and clinical features (Figures 1.1 and 1.2).
Lymphomagenesis in these disorders are driven by genetic alterations, allowing escape from the normal physiological restrictions on growth, differentiation, proliferation and cell death, resulting in a suite of genetic lesions that differ not only between various B-NHL subtypes, but also from case to case within each individual subtype. The underlying molecular heterogeneity of B-NHL is suggested by the highly variable clinical behaviour of different lymphoma subtypes and the variation in the susceptibility to various molecularly targeted and novel therapies (Blombery et al., 2015).
This thesis addresses the processes of progression and transformation in two of the most common subtypes of mature B-cell neoplasms: follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). Both of these could be considered indolent, and patients do not need to receive treatment until they manifest symptoms (the “watch and wait” strategy).
However, a fraction of patients of both types progress or transform to exhibit a more aggressive disease, which shortens their life expectancy dramatically.
The B-NHL subtypes and their incidence are specified in Figure 1.2 These subtypes can be classified into two groups: low-grade and high-grade.
In the low-grade subgroup, the most common subtypes are FL and CLL; in the high-grade subgroup, DLBCL is the most common type of B-NHL.
Figure 1.2. Incidence of B cell non-Hodgkin lymphomas.
B-Cell Chronic Lymphoid Leukaemia/Small Lymphocytic Lymphoma
General Features
CLL is characterized by clonal proliferation and the accumulation of mature CD5+ CD23+ B lymphocytes that could infiltrate the bone marrow, peripheral blood, lymph node and spleen (Zenz et al., 2010). CLL is the most prevalent leukaemia in Western countries, and is characterized by a variable clinical course, whereby some patients never require therapy and others display an aggressive course, with a poor response to therapy and death within months.
It is mainly a disease of the elderly, with nearly double the incidence in men as in women.
Pathology
The leukaemia cells found in the blood are small mature lymphocytes with a narrow border of cytoplasm and a dense nucleus that lacks discernible nucleoli and with partially aggregated chromatin. These cells may be admixed with larger or atypical cells, cleaved cells or
CLL cells co-express the T-cell antigen CD5 and B-cell surface antigens CD19, CD20, and CD23.
The levels of the surface immunoglobulins Ig-CD20 and Ig-CD79b are characteristically low compared with those found on normal B cells (Ginaldi et al., 1998, 1998; Moreau et al., 1997).
Each clone of leukaemia cells are restricted to the expression of either kappa or lambda immunoglobulin light chains (Ginaldi et al., 1998).
The survival of CLL cells also depends on a permissive microenvironment of cellular components. Macrophages, T cells, and stromal follicular dendritic cells stimulate crucial survival and pro-proliferative signalling pathways in leukemic cells by secreting chemokines, cytokines and angiogenic factors, or by expressing distinct surface receptors or adhesion molecules (Burger and Gribben, 2014).
Clinical Features
CLL is more common in men than in women (with a ratio of 1.5-2:1) and more common in white than in black ethnicities. It is an uncommon malignancy in Asia. The etiological factors for typical CLL are unknown (“WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Fourth Edition - WHO - OMS -,” 2018). The median age at diagnosis is approximately 70 years, but the disease can also be present in younger adults (Parikh et al., 2014).
A limited number of risk factors for CLL have been identified, but it has one of the strongest inherited predispositions of all haematological malignancies. About 10% of individuals who develop CLL have a family history of the disease (Cerhan and Slager, 2015).
CLL involves the blood, bone marrow, and secondary lymphoid tissues such as the spleen, lymph nodes, and the Waldeyer ring. Extra-nodal involvement (skin, gastrointestinal tract, or CNS) occurs in a small subset of cases (Ratterman et al., 2014).
Prognostic Markers
Classical Staging Systems
There are two accepted, complementary clinical staging systems: those of Rai (Rai et al., 1975) and Binet (Binet et al., 1981). Both groups describe three major prognostic groups with
discrete clinical outcomes. Both systems are simple and inexpensive, relying on a physical examination and standard laboratory tests.
The Rai staging system is based on lymphocytosis:
Low Risk. Stage 0: Lymphocytosis with leukaemia cells in the blood and/or bone marrow (lymphoid cells >30%)
Intermediate Risk. Stage I and stage II: Lymphocytosis, enlarged nodes at any site, and splenomegaly and/or hepatomegaly
High Risk. Stage III: Disease-related anaemia (haemoglobin level (Hb) < 11g/dl) and thrombocytopenia (platelet count of < 100 x 109/L) stage IV.
The Binet staging system is based on the number of involved areas of nodal or organ enlargement, and on whether there is anaemia or thrombocytopenia.
Binet A. Up to two areas involved with no anaemia or thrombocytopenia (Hb ≥10g/dL and platelets ≥ 100 x 109/L).
Binet B. Three or more areas involved with no anaemia or thrombocytopenia (Hb ≥10g/dL and platelets ≥ 100 x 109/L).
Binet C. Anaemia and thrombocytopenia (Hb < 10g/dL and platelets < 100 x 109/L).
IGHV Status
There are two CLL subgroups defined by the mutational status of IGHV genes: the IGHV mutated status (IGHV-M, mutations detected at a level of 2% or higher in the sequenced V region of the clonal rearrangement) is associated with long-lasting stable disease and good prognosis, probably because of different genetic and/or epigenetic alterations, activated signalling pathways or microenvironment interactions. Conversely, the IGHV un-mutated genotype (IGHV-U) is associated with a more active and proliferative disease, partly as a consequence of its association with concurrent genomic mutations that adversely effect on clinical outcome (Fabbri and Dalla-Favera, 2016; ten Hacken and Burger, 2016; Vardi et al., 2014)
Immunophenotypic Markers
CD38 is a transmembrane glycoprotein widely expressed in a range of haematopoietic and non-haematopoietic cell types. The high level of expression of CD38 in leukaemic lymphocytes (≥ 30% of neoplastic cells) is associated with poor overall survival (OS) in patients with CLL
ZAP70 is a member of the Syk-ZAP-70 protein kinase family. It is normally expressed in T and natural killer cells and has a critical role in the initiation of T-cell signalling (Chan et al., 1992).
The role that ZAP-70 expression plays as an independent prognostic marker in CLL and its association with IGHV mutational status are controversial (Chan et al., 1992; Dürig et al., 2003;
Liu et al., 2018).
Recently, CD49d has been reported to be an unfavourable prognostic marker in CLL. Patients with ≥ 30% of neoplastic cells expressing CD49d were considered positive. The decrease in OS at 5 and 10 years among CD49+ patients was 7% and 23%, respectively (Bulian et al., 2014;
Dal Bo et al., 2016).
Cytogenetics
About 80-90% of cases have cytogenetic abnormalities, as detected by fluorescence in situ hybridization (FISH) or conventional G-banding cytogenetics. The most frequent chromosomal aberrations are deletions in 13q14.3 (present in approximately 50% of CLL patients) and trisomy of chromosome 12, or partial trisomy 12q13 (present in 10-20%). Less commonly, there is a deletion in 11q22-23 (present in 10% of cases), 17p13 (TP53, present in 5-8% of cases), or 6q21 (7%). All deletions of 11q cause the loss of the ATM gene, which encodes the DNA damage response kinase ATM, and some of them also involve the BIRC3 gene. More than 80% of cases with deletion in 17p also carry mutations in the other TP53 allele, resulting in the functional disruption of the TP53 pathway. Patients with 11q and 17p deletions have poor survival compared with those with trisomy 12 or a normal karyotype, or patients with 13q deletion as their sole abnormality (Döhner et al., 2000). Recurrent genomic aberrations play an essential role in aiding prognosis in CLL and in guiding therapy.
New Staging Score
A prognostic score has recently been developed by an international consortium of study groups called the “CLL International Prognostic Index Group” (International CLL-IPI working group, 2016). This group has identified five independent prognostic factors:
- Age > 65 years old
- TP53 deletion and/or mutation - IGHV mutational status
- Serum β2-microglobulin > 3.5 - Clinical stage (Binet and Rai)
Using a weighted score of the five independent factors they have identified four risk groups with different patterns of survival. They have also proposed a treatment approach for each risk group (Table 1.1).
CLL-IPI category OS at 5
years (%) Probable clinical recomendation
Low risk 93.2 Do not treat
Intermediate
risk 79.3 Do not treat unless the disease is genuinely symptomatic High risk 63.3 Treatment indicated unless the disease is asymptomatic
Very high risk 23.3 If you need to treat, do not use chemotherapies, but instead use novel agents or treatment in clinical trials
Table 1.1 Different CLL-IPI categories. OS of each CLL-IP category and probable clinical recommendation.
Genetic Landscape
In the last years, various studies have mapped out the genetics of CLL using next-generation sequencing to reveal the genetic landscape of somatic mutations. The most commonly mutated genes, affecting 3-15% of cases, are TP53 (7-15%), SF3B1 (9-15%), NOTCH1 (4-15%), MYD88 (1-3%), BIRC3 (2.7-5%), POT1 (4.8%) and ATM (8-12%). The diversity of results is affected by the different cohorts, from pre-treated to relapsed or progressed and the development of the techniques that allow alleles with a frequency of less than 10% to be detected (Fabbri et al., 2011; Fabbri and Dalla-Favera, 2016; Landau et al., 2013; Ljungström et al., 2016; Quesada et al., 2011; Wang et al., 2011). High frequencies of mutations are linked to poor outcome and aberrations of some genes. In particular, TP53, NOTCH1 and SF3B1 are more frequent at relapse. Retrospective studies in untreated patients from historical cohorts have recently shown the adverse prognostic impact of TP53, NOTCH1 and SF3B1 mutations on the time to treatment and OS (Baliakas et al., 2015; Jeromin et al., 2014; Rossi et al., 2013).
Minimal Residual Disease in CLL
The assessment of minimal residual disease (MRD) has been introduced as an additional and increasingly important parameter of response assessment. It is an important endpoint in the treatment of CLL, being highly predictive of prolonged progression-free survival (PFS) and OS.
MRD can be assessed by flow cytometry (4-colour or 6-colour flow cytometry, polymerase chain reaction (consensus PCR, nested clone-specific PCR or allele-specific oligonucleotide (ASO) IGHV PCR) and high-throughput sequencing, which is the most sensitive and cutting- edge technology currently available (Thompson et al., 2018).
At present, MRD is a very promising tool for estimating prognosis and for influencing decision- making during the course of treatment. The challenge now is to standardize it across centres and to validate MRD as an endpoint in a representative population (Thompson et al., 2018).
Follicular Lymphoma
General Features
Follicular lymphoma (FL) is a neoplasm derived from germinal centre B cells (typically centrocytes and centroblasts/large transformed cells), which usually have at least a partially follicular pattern.
FL is the most common indolent NHL in western countries and accounts for 22-25% of NHLs according to the World Health Organization (“WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, Fourth Edition - WHO - OMS -,” 2018). Although FL is considered incurable by standard chemotherapy, advances in treatment and our understanding of its biology have improved the management of this disease and its clinical outcomes. The natural history of FL appears to have been modified to the benefit of patients by the introduction of rituximab (Kahl and Yang, 2016).
FL predominantly involves the lymph nodes, but also the spleen, bone marrow, peripheral blood and, less commonly, the Waldeyer ring. Any nodal site can be involved, but most patients present with peripheral lymphadenopathy.
There are several unmet needs, including a better ability to identify high-risk patients at diagnosis, the development of predictive biomarkers for targeted agents, and strategies to reduce the risk of transformation (Kahl and Yang, 2016).
Clinical Features
The European incidence of FL is 2.18 per 100,000 inhabitants (Sant et al., 2010). The incidence varies markedly between Western and Asian countries, ranging from 3.8-12% in Asian
There is a sex imbalance in FL, with males predominating in all population-based studies (Biagi and Seymour, 2002). The median age at diagnosis is 60-65 years (“A clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin’s lymphoma. The Non- Hodgkin’s Lymphoma Classification Project,” 1997; Freedman, 2018). Most patients have widespread disease at diagnosis, including peripheral and central (abdominal and thoracic) lymphadenopathy and splenomegaly. The bone marrow is involved in 40-70% of cases. Only 15-25% of cases are stage I or II at the time of diagnosis (Hiddemann and Cheson, 2014).
Patients are usually asymptomatic and B symptoms, such as fever and weight loss, are uncommon. Transformation, mainly to DLBCL, occurs in 25-70% of patients (Freedman, 2018;
Wagner-Johnston et al., 2015). In FL, a watch-and-wait strategy, or a strictly palliative approach, is often more appropriate than an aggressive therapy (there are no differences in OS between the different approaches) (Kahl and Yang, 2016). For symptomatic patients with a high tumour burden the most commonly used regimens are R-CHOP, R-CVP and R- fludarabine (Kahl and Yang, 2016).
Staging score
The FL International Prognostic Index (FLIPI) includes 5 prognostic factors patient age, stage, number of involved nodal areas, serum lactate dehydrogenase, and haemoglobin, groups in table 1.2 are stablished according to the following values:
- Age > 60 Serum - LDH > ULN
- Haemoglobin < 12 g/dl - Stage III or IV
- Number of nodal sites > 4
Risk group # Risk factors 2 year OS 2 year PFS
Low risk 0–1 98% 84%
Intermediate risk 2 94% 70%
High risk 3 or more 87% 42%
Table 1.2 Follicular Lymphoma International Prognostic Index risk groups.
Pathology
FL is derived from germinal centre (GC) B cells. Its pathogenesis is closely linked to the normal GC reaction in which naïve B cells from the bone marrow undergo somatic hypermutation (SHM) and class-switching recombination of the BCR in a process that generates immunoglobulin diversity and selects B cells producing high-affinity antibodies (Basso and Dalla-Favera, 2015; MacLennan, 1994). These tumours contain a mixture of neoplastic centrocytes and centroblasts along with various non-neoplastic cells, including T cells, follicular dendritic cells (FDCs), and macrophages, and mirroring the formation of secondary lymphoid follicles. Progression of cytological grade is common during the natural course of the disease. A diffuse pattern composed of centroblasts is considered evidence of progression to diffuse-large B cell lymphoma (DLBCL) (“The 2008 WHO classification of lymphoid neoplasms and beyond: evolving concepts and practical applications. - PubMed - NCBI,” 2018)
In FL tumours, malignant cells continue to be substantially dependent on microenvironmental cells for survival and proliferation signals. Tumoral cells may be accompanied by FDCs, T helper cells, macrophages, benign B cells, endothelial cells and an extracellular matrix, depending on the stage of the disease (Scott and Gascoyne, 2014).
Figure 1.3 Follicular lymphoma Pathogenesis. Naive B cells in the bone marrow (BM) acquire the t(14;18) translocation due an error in V(D)J recombination and subsequently home to B-cell follicles where they undergo the GC reaction. In the dark zone of the GC, the B cells proliferate as centroblasts and undergo somatic hypermutation (SHM) and class switching of their BCRs. Centroblasts then become smaller centrocytes and migrate to the light zone of the GC where they interact with follicular dendritic cells (FDCs) and are selected to either undergo apoptosis or rescue by follicular helper T cells (TFH) based on antigen (Ag) affinity of their BCRs. Ectopic expression of BCL2 provides mutant B cells with t(14;18) an avenue to escape apoptosis, independent of BCR affinity. These FL-like B cells then exit the GC and enter the circulation where they might be prone to traffic between follicles and/or the BM and can acquire additional genetic changes necessary for transformation to FL.
Genetics and Cytogenetics
The t(14;18) translocation is found in 85% of FLs and places the BCL2 gene under IGH regulatory elements. Although dysregulation of BCL2 expression is not sufficient to induce lymphomagenesis, it confers a survival advantage through the activation of antiapoptotic programs that are typically repressed by BCL6 in GC B cells (Kahl and Yang, 2016; Roulland et al., 2006). Expression of activation-induced deaminase (AID) in normal GCs results in SHM of immunoglobulin loci (Figure 1.4). AID is expressed in the precursor FL cells as they reside in germinal centres in lymph nodes, it produces mutations in some genes (SGK1, BCL2, PIM1,
Next-generation sequencing studies have identified chromatin-modifying gene (CMG) mutations as a hallmark of FL. Recent studies have provided insight into the functional consequences of the most frequently mutated CMGs (KMT2D, CREBBP, EP300 and EZH2 accounting for ~33%, 27%, 15%, and 9% of FL, respectively) and suggest a role for these events in modifying normal B-cell differentiation programs and impeding germinal centre exit. The inactivating mutations of KMT2D interfere with the ability of KMT2D to activate gene transcription through H3K4 methylation (Okosun et al., 2014). However, the majority of FL tumours serially acquire multiple CMG mutations, suggesting that there is a level of cross-talk or cooperation between these events that has not yet been identified (Morin et al., 2011;
Okosun et al., 2014; Pasqualucci et al., 2011, 2014) .
Follicular Lymphoma Transformation
General Features
Histological transformation (HT) refers to a biological event leading to the development of a high-grade, aggressive NHL in patients with an underlying FL (Cullen et al., 1979).
Transformation most commonly results in a histology that cannot be distinguished from de novo DLBCL, although the histology of some patients bears some resemblance to Burkitt lymphoma (De Jong et al., 1988; Kridel et al., 2015; Lossos and Gascoyne, 2011; Montoto and Fitzgibbon, 2011; Swerdlow et al., 2016).
HT is associated with rapid progression, treatment resistance, and poor prognosis. Recent modifications to the physiopathological mechanism of transformed FL (t-FL) have been proposed, including genetic and epigenetic mechanisms and a role for the microenvironment.
Although t-FL is considered a devastating complication, because it is associated with treatment-refractory disease and a dismal outcome (Fischer et al., 2018), a recent report from the US National LymphoCare Study evaluated outcomes in 2652 patients and found the risk of transformation remained at 2-3% per year in the R-chemo era. The risk was similar to that faced by R-CHOP and R-CVP treated patients, suggesting that there is no reduction in risk associated with the upfront inclusion of anthracyclines. However, the risk was reduced in patients receiving maintenance rituximab, the median OS after transformation was 5 years, which was markedly better than values in historical reports (Wagner-Johnston et al., 2015).
Prior to the use of rituximab, median OS of patients with HT was very poor, approximately 1
to 2 years across several series (Montoto and Fitzgibbon, 2011). Immunotherapy has produced a significant reduction in the 10-year cumulative hazard of histological transformation of just 5.2 in patients who received rituximab compared with 8.7% in those who did not (Federico et al., 2018).
It has been suggested that several distinct mechanisms are likely to be involved in transformation, including alterations of cell-cycle control (through mutation or deletion of cyclin-dependent kinase 2A/B [CDKN2A/B] and alterations in MYC) and impairment of the DNA damage response (through loss of TP53 and/or CDKN2A) (García-Sanz et al., 1998; Pinyol et al., 1998). Furthermore, there are consistent losses of genes associated with regulation of the immune response, such as the entire HLA class 1 locus, mutations specifically in B-2 microglobulin (B2M), and mutations in CD58, which is involved in regulating the complement- mediated effects on cells (Bouska et al., 2014; Casulo et al., 2015; Challa-Malladi et al., 2011).
Genetics and Cytogenetics
Since 2014, while the work presented in this thesis was being carried out, some research groups have made an effort to elucidate the molecular mechanisms involved in transformation. The first attempts studied small cohorts of up to 12 patients (Okosun et al., 2014; Pasqualucci et al., 2014); reports from bigger cohorts have been published in the last two years (Kridel et al., 2017; Krysiak et al., 2017).
The most common aberration specifically acquired during progression to t-FL was the loss of CDKN2A/B, two tumour suppressor genes whose protein products play a major role as negative regulators of cell-cycle G1 progression and as stabilizers of the tumour suppressor p53. CDKN2A/B biallelic lesions tend to be mutually exclusive with biallelic deletions and/or mutations of TP53 (Kridel et al., 2017; Okosun et al., 2014; Pasqualucci et al., 2014). Genetic lesions have been found that deregulate MYC (translocations, copy number gains and/or amplifications), giving rise to a loss of proliferation control when the expression is increased, and a loss of the apoptotic mechanism associated with decreased c-myc expression (Lossos et al., 2002), B2M and CD58 biallelic mutations and/or deletions, genes involved in the control of immune recognition by cytotoxic T lymphocytes and NK cells (Pasqualucci et al., 2014).
and B cell development pathways (Bouska et al., 2014; Kridel et al., 2017; Okosun et al., 2014;
Pasqualucci et al., 2014).
Diffuse Large B Cell Lymphoma Transformed from FL
DLBCL is the most prevalent B-NHL in adults, comprising 30-40% of all new diagnoses and including cases that arise de novo and cases that result from the histological transformation of various, less aggressive B-NHL types (i.e., FL and CLL) (Campo et al., 2011).
The current 5-year OS rate is 60–70% for DLBCL patients, while patients with t-FL have a life expectancy of 2.5 months to 2 years (Chaganti et al., 2016; Martelli et al., 2013; Perry et al., 2016; Teras et al., 2016; Yao et al., 2018).
DLBCL is a heterogeneous entity in terms of clinical presentation, genetic findings, response to therapy, and prognosis. The application of gene expression profiling (GEP) to the study of DLBCL was a major advance that clarified this heterogeneity and provided a rationale for subdividing cases into groups (Alizadeh et al., 2000; Li et al., 2018; Scott et al., 2014).
Classification by cell-of-origin (COO) divides DLBCL into two subtypes: germinal centre B cell- like (GCB) and activated B cell-like (ABC) subtypes. About 10-15% of cases are unclassifiable (Scott et al., 2014). Patients with the GCB subtype usually have better prognosis than those with the ABC subtype (Li et al., 2018; Scott et al., 2014). Although t-FLs could be included in the GCB subtype on the basis of their histological and genetic features, their prognosis is poor after transformation.
Rearrangement of the 3q27 region involving BCL6 occurs in 30% of cases. It is the most common translocation in DLBCL and is critical for GC formation. This translocation in t-FL is slightly less common (~20%). The t(14;18) translocation occurs in 20-30% of patients and leads to overexpression of the BCL2 gene found on chromosome 18. The MYC rearrangement is observed in 8-14% of cases. Some other patients who do not have the translocation also overexpress the BCL-2 protein (Longo, 2015). Approximately half of the DLCBL cases that harbour a MYC translocation also show a BCL2 and/or BCL6 translocation, are newly classified as high-grade B cell lymphoma, and are known as “double-hit” lymphomas (DHLs), or “triple- hit” lymphomas (Rosenthal and Younes, 2017; Swerdlow et al., 2016).
The recurrently mutated genes of transformed FL resemble that of GCB DLBCL. The most
frequently mutated genes of this subtype are: TNFSF14, CREBBP, B2M, BCL2, SOCS1, EZH2, SGK1, IRF8 and GNA13, amongst others. This mutational profile bears a similarity to the FL mutational profile (Chapuy et al., 2018; Schmitz et al., 2018) and differs from the ABC group in which the most recurrently mutated genes are CDKN2A, PIM1, MYD88, CD79B, PRDM1 and CD58 (Schmitz et al., 2018) while in GCB subtype are EZH2, CREPPB, TET2 and IDH2.
Tumour Heterogeneity and Clonal Evolution
In 1976, Peter Nowell published a landmark perspective on cancer as an evolutionary process that is driven by stepwise, somatic cell mutations with sequential, subclonal selection (Greaves and Maley, 2012; Nowell, 1976). Tumours evolve through competition and interactions between genetically diverse clones. This process has been postulated as being parallel to Darwinian natural selection, together with natural selection of the fittest variants.
Clones evolve through the interaction of selectively advantageous driver lesions, selectively neutral passenger lesions and deleterious lesions (Greaves and Maley, 2012).
Tumour heterogeneity is defined as the presence of subpopulations of cells possessing different genomic alterations within a tumour (Aparicio and Caldas, 2013) and has been associated with tumour development (Landau et al., 2013) invasion and metastasis (Campbell et al., 2010), and poor clinical outcome (Bochtler et al., 2013). Greater access to deep- sequencing techniques is extending our knowledge about the prevalence of intra- and inter- tumoral heterogeneity (Fisher et al., 2013).
The clonal evolution model posits that, after transformation of a single founding neoplastic cell, tumours evolve through an iterative and dynamic process as they continuously accumulate somatic alterations, some of which confer selective growth advantages. In the classic linear progression model, stringent positive selection for phenotypic traits results in selective sweeps in the course of tumour progression (Greaves and Maley, 2012; Nowell, 1976; Scott and Marusyk, 2017)
Most appropriately interrogated cancers are found to have intra-clonal genetic heterogeneity that differs from the pre- and post-transformation tumour, which may indicate divergent clonal evolution. Clonal architecture might be driven by genetic heterogeneity of propagating or “stem” cells (Greaves, 2010)
Detailed characterization of clonal dynamics should reveal fundamental biological properties, and have implications for future patient management strategies with respect to transformation, progression and response to treatment.
Haematological malignancies have demonstrated not only a high degree of clonal heterogeneity and marked changes in the genetic makeup of the disease upon release, but also a predominant pattern of branching rather than linear evolution (Ding et al., 2012; Landau et al., 2013; Welch et al., 2012).
Figure 1.4 Patterns of clonal evolution in cancer.
CLL and FL are two ideal models for studying tumour heterogeneity. They are both slow- growing lymphomas with a long median OS, which enables the collection of successive samples with which to elucidate the evolution of clonal composition.
Transformation of FL has been described mainly as a process of divergent branched evolution from a common progenitor cell, while transformation and progression in CLL arise without significant branching, instead mimicking a pattern of linear evolution.
Clonal evolution is a key mechanism in the development of treatment resistance and refractoriness in leukaemia and lymphoma (Landau et al., 2013, 2015; Puente et al., 2011).
Resistance to treatment can be classified as primary, describing patients who exhibit no response to treatment at all, or secondary, for patients who initially respond to treatment, but later develop resistance by the selection or appearance of a clone, whereby the cells that are sensitive to treatment die and the resistant cell population continues to grow (Pogrebniak and Curtis, 2018). Exposures to treatments change cancer–clone dynamics by introducing a potent source of artificial selection in the form of drugs or radiation, but evolutionary principles still apply. Usually, treatment results in massive cell death, which imposes a selective pressure for the proliferation of variant cells that resist treatment, and this changes the clonal composition and disrupts the clonal equilibrium. Furthermore, many cancer therapeutic agents are genotoxic; cells that survive the treatment, which could then go on to regenerate the cancer, may gain further mutations, giving rise to cells with greater fitness and malignant potential (Greaves and Maley, 2012).
The ability of cancer cells to evolve and adapt to targeted therapies presents a challenge that limits the success of treatments and the durability of responses.
Genomics: Next-Generation Sequencing
Since 2008, “next-generation sequencing” has become increasingly accessible to researchers.
The application of genomic tools to human cancer has been an exceptionally active area of research and development since the earliest days of research in this field. The availability of these techniques has greatly advanced our knowledge and understanding of lymphoma genetics, enabling us to describe the mutational landscape of the different types of lymphoma and leukaemia. It has also provided further insight into the complexity of the genetics and evolutionary biology of cancer cells (Stratton, 2011).
Next generation sequencing (NGS) has enabled the detection of low-frequency mutations (with allele frequencies less than 10%). Subclonal mutations insome genes (e.g., TP53) have been shown to have the same consequences for the patient as higher-frequency mutations (Nadeu et al., 2016). However, considerable work is still needed to determine the prognostic implications and therapeutic effects of the mutations identified.
Understanding the genetic changes and gene expression profiles of cancer cells is leading to more effective treatment strategies that are tailored to the genetic profile of each individual patient’s cancer. Target genes have already been discovered that, when mutated, make the tumour susceptible/sensitive to specific therapies (e.g., crizotinib in ALK-positive patients).
The challenge now is to construct a comprehensive catalogue of key genomic changes and their evolution along the disease, that allow more effective and specific treatments for each patient (Verma, 2012).
HYPOTHESES AND OBJECTIVES
2. HYPOTHESES AND OBJECTIVES
This work presented in thesis addresses clonal heterogeneity in lymphoid neoplasms through two projects that focus on the most common lymphoproliferative low-grade B cell malignancies, CLL and FL, and the processes involved in their progression and transformation.
Project I: “Clonal evolution and dynamics involved in the progression of chronic lymphocytic leukaemia”
• Part A. “Genomic mutation profile in progressive chronic lymphocytic leukaemia patients prior to first-line chemoimmunotherapy: predictive outcomes from the REM Clinical Trial”
• Part B. “Monitoring clonal dynamics during clinical evolution in chronic lymphocytic leukaemia”
Project II: “Unravelling the transformation from follicular lymphoma to diffuse large B-cell lymphoma”
Project I
CLL is the most common leukaemia in adults in western countries and remains an incurable disease. Studies of CLL genetics have revealed a high degree of molecular heterogeneity in these tumours. The challenge now is to understand their complex genetic landscape and the association between clinical phenotypes, response to therapies and long-term outcomes.
Studying the evolution of cancer cells during disease progression and under therapeutic selective pressure would be a crucial step towards understanding the mechanisms that lead to treatment resistance.
Taking this into account, the aims were:
1. To identify predictive markers of progressive CLL patients with respect to first-line chemoimmunotherapy.
2. To elucidate the molecular mechanisms and clonal heterogeneity and evolution involved in progression and treatment response.
3. To identify the subclonal composition and molecular differences between the various organs affected by CLL.
Project II
FL is an indolent, yet incurable B cell malignancy, and a subset of patients undergo histological transformation to a more aggressive B cell neoplasm (the most common DLBCL), which leads to a poorer clinical outcome. An understanding of the clonal dynamics during disease transformation and of the genetic events occurring at particular times would yield information that could be used to provide better monitoring and more effective targeting of these tumours.
1. To elucidate the genetic alterations involved in the transformation of FL, and to identify new therapeutic approaches.
2. To identify biological predictive markers to predict transformation at the time of FL diagnosis.
MATERIALS AND METHODS
3. MATERIALS AND METHODS Patients and Cell Lines
The three projects were approved by the Hospital Universitario Puerta de Hierro- Majadahonda institutional Ethical Committee. Patients provided informed consent in accordance with local institutional review board requirements and the Declaration of Helsinki before enrolment. All research methods were performed in accordance with Institutional Guidelines and regulations. Treatment protocols were according to the internal clinical services guidelines.
Patients and samples - Project Ia
Ninety peripheral blood samples from treatment-naïve CLL patients with progressive active disease were included in the study. Seventy-one patients were enrolled in the REM (Rituximab in maintenance) clinical trial. REM is a multicenter non-randomized prospective phase II clinical trial to evaluate the overall response and PFS in CLL patients with active progressive disease, after first line treatment with FCR followed by Rituximab maintenance every two months for three years in responding patients (Garcia-Marco et al. 2018, submitted). The remaining 19 patients were selected based on similar clinical course and therapeutic regimens (FCR; one patient regimen also included mitoxantrone); rituximab plus bendamustine or cyclophosphamide, mitoxantrone and fludarabine). All samples were collected before treatment.
REM study was registered as clinical Trial with NCT#: 00545714 and EudraCT#: 2007-002733- 36.
Patients and samples - Project Ib
Samples from two CLL patients collected during disease evolution and from different body locations were analyzed.
Patient 1, a physically fit 50-year-old male, was diagnosed in September 2000 with stage A/II CLL, with splenomegaly of 4 cm below the left costal margin (b.l.c.m.), a lymphocyte count of 112,000/µL, a haemoglobin count of 14.5 g/dl, and a platelet count of 150,000/µL. He was
23 del (ATM del in 67% of interphase nuclei), as revealed by fluorescence in situ hybridization (FISH).
In May 2004, 45 months after diagnosis, the patient presented with increasing fatigue, a lymphocyte doubling time of 6 months and splenomegaly of 6 cm b.l.c.m. He was treated with fludarabine (25 mg/m2 x 5 days x 6 cycles) and achieved partial remission.
He presented at 21 months since first treatment (64 months from diagnosis) with symptomatic disease, generalized lymphadenopathy and splenomegaly of 4 cm b.l.c.m. (Sample P1.1, February 2006) and in March 2006 (67 months from diagnosis) was treated with FCR (fludarabine, cyclophosphamide, rituximab) plus rituximab maintenance for 6 months, achieving complete clinical remission, and was consolidated with a non-myeloablative allogeneic stem cell transplant (SCT) of HLA-identical unrelated adult donor in June 2007 (82 months after diagnosis) (Sample P1.2, before transplant).
The patient relapsed 20 months after transplantation (102 months, February 2009) with rapidly progressive lymphocytosis and generalized lymphadenopathy of 2-4 cm and retroperitoneal adenopathy of 6 cm, along with acquisition of a 17p1.3 deletion, as revealed by FISH, and a TP53 mutation, as identified by cDNA Sanger sequencing (inframe deletion:
c.376_396del21; p.Y126_K132delYSPALNK). He was treated with rituximab and increasing monthly doses of donor lymphocyte infusion (4 cycles), and achieved partial remission (PR).
The patient progressed in October 2010, developing rapidly growing generalised lymph nodes and lymphocytosis that were refractory to two subsequent lines of treatment (R- bendamustine and R-lenalidomide).
In February 2011 (126 months after diagnosis), the patient received four cycles of salvage chemotherapy with dexamethasone, high-dose cytarabine and oxaliplatin, achieving PR, and was consolidated in August 2011 with a second non-myeloablative allogeneic SCT of HLA- identical unrelated cord-blood. Patient remained in partial remission until February 2012 (138 months after diagnosis), when progressed with a 7-cm bulky retroperitoneal lymphadenopathy and was treated with ofatumumab-bendamustine for 3 cycles, but without clinical response. In September 2012 the patient died of disease progression without histopathological evidence of Richter transformation.
Patient 2, a physically fit 62-year-old male, was diagnosed in September 2006 with stage B/II CLL with generalized lymphadenopathy of 1.5-2.0 cm without hepatomegaly or splenomegaly,
a lymphocyte count of 16.750/µL, a haemoglobin count of 14.8 g/dl and a platelet count of 159,000/µL. His sample was CD38-negative, ZAP-70-positive, IgHV unmutated, and had a normal karyotype with deletions of 11q22-23 (ATM) and 13q14 (D13S319) detected by FISH in 50% and 25% of interphase nuclei, respectively.
The patient was treated in March 2008 (15 months after diagnosis) with 6 cycles of FCR, and rituximab maintenance for 3 years (REM clinical trial), and he achieved a complete response.
In October 2012 (70 months after diagnosis) the patient developed rapidly increasing lymphocytosis with generalized lymphadenopathy of 3-4 cm and molecular cytogenetic studies showed a complex acquired karyotype [45-46, XY, -10, del(11)(q14;q21), del(11)(q11), del(13)(q14;q21), i(17)(q10)] with deletions of 17p13 (p53) (55%), 13q14 (63%), C-MYC amplification (19%) and a notably low percentage of ATM deletion (9%) in the 11q22-23 region. The patient was treated with anti-CD37 plus bendamustine for 6 cycles (clinical trial) and achieved partial remission. In July 2013 (79 months) presented with rapidly growing cervical and axillary lymphadenopathy of 3-5 cm along with progressive malaise, weight loss, night sweats and fever with no documented active infection. A lymph node biopsy showed Richter transformation into a diffuse large B cell lymphoma (DLBCL) (Ki67: 70%, CD20+ high, CD5-, CD10-, BCL-6-, MUM-1+, FOXp1+, CD38+ and EBERs-). The bone marrow had no infiltration by DLBCL but showed diffuse infiltration of small and medium size lymphocytes.
CD20+, CD5+, CD23+, BCL2+ and TP53 overexpression and Ki67+(40%). FISH data showed 17p13 deletion (75%), homozygous 13q14 deletion (83%), and CMYC amplification/translocation (19%). P2 was treated with salvage immunochemotherapy (rituximab, liposomal doxorubicin and prednisone) and bortezomib for 2 cycles without clinical response and died in January 2014 (86 months after diagnosis), 7 months after diagnosis of transformation to non-germinal center DLBCL with the same IGHV mutational pattern as the original CLL clone.
Fifteen samples were collected from peripheral blood of Patient 1 between diagnosis and exitus (P1.1 to P1.13) as well as biopsies from lymph node (P1.14) and spleen (P1.15) from necropsy and buccal mucosa (P1.N). We analysed three samples from peripheral blood
Patient 2 also participated in two research phase II clinical trials (REM: EudraCT No.: 2007- 002733-36 and Otlertuzumab plus Bendamustine: NCT01188681) approved by the Institutional ethics committee.
Patients and samples– Project II
Paired formalin-fixed, paraffin-embedded tissue (FFPET) samples, consisting of paired FL (pre- tFL) and FL transformed to DLBCL (tFL) from 22 transformed patients and the FFPET diagnostic samples from 20 non-transformed FL (ntFL), were collected. We obtained the germinal DNA of eight patients from oral mucosa or other non-neoplastic biopsies. Samples and clinical data were collected (Figure 3.1 and Table S1 in CD), processed and stored according to quality protocols, ensuring the safety and confidentiality of donors’ data. All samples were reviewed by expert hematopathologists upon arrival to confirm their diagnosis and to select enriched tumoral areas as necessary.
Patient selection was made according to:
- Diagnosis of FL grade 1-3A (we excluded grade 3B)
- Minimum follow up of seven years for non-transformed patients - Histological confirmation of transformation for transformed patients - Availability of paired samples (pre-tFL and tFL) for transformed patients
An additional cohort of 48 samples from patients with de novo DLBCL including GCB and ABC type cases, (data not shown) was included to enable comparison.
Figure 3.1 Temporal evolution of transformed patients. pre-tFL biopsies: black circles; tFL biopsies: grey squares.
Cell lines and culture conditions – Project II
The human tumoral cell lines used in the study are summarized in Table 3.1 The cell lines were kindly supplied by the research group of cancer genomics at IDIVAL (Santander, Spain). Cell lines were grown in RPMI 1640 medium supplemented with 2mM Glutamine (HyClone, GE Healthcare Life Sciences, Illinois, United States) and 10% heat-inactivated fetal bovine serum (FBS) (BioWest, Nuaillé, France) and 1% penicillin-streptpomycin (HyClone, GE Healthcare Life Sciences, Illinois United States). Cell lines were authenticated at the genomics unit at “Instituto de Investigaciones Biomedicas Alberto Solls”. Cell lines were counted with Accuchip kit for ADAM-MC Automatic Cell Counter (NanoEnTek, Seoul, Korea).
Cell Line Lymphoma type Growth medium Growth
RAJI Burkitt Lymphoma RPMI + 10% FBS Suspension
RL DLBCL RPMI + 10% FBS Suspension
SUDHL-6 DLBCL RPMI + 10% FBS Suspension
OCILY-19 DLBCL RPMI + 10% FBS Suspension
Table 3.1 Description of the cell lines used in the study.
Clinical, Molecular and Pathological data
Clinical data
Patients’ data were collected by the assigned physicians of each hospital (collaborative centres are listed in Annex I) and centralized at Hospital Universitario Puerta de Hierro-Majadahonda using RedCap platform (Vanderbilt University Medical Center, Tennessee, USA). Clinical data was collected, processed and stored according to quality protocols, ensuring the safety and confidentiality of donors’ data.
Project I
The following clinical and laboratory data were collected:
- Gender - Age
- Date of diagnosis
- Stage at time of treatment - Binet Stage
- Rai Stage
- Date of first line treatment - First line treatment
- Date of clinical response evaluations - Date of second line treatment - Second line treatment
- Date of Progression - Patient status - Date of death - Cause of death
- Date of last visit
- MRD (Bone Marrow and Peripheral Blood) at the end of treatment
Project II
The following clinical and laboratory data have been collected for this project:
- Gender - Age
- Dates of diagnosis and transformation - Other tumors
- Diagnosis of FL and diagnosis of HT:
o Grade
o Origin (nodal or extra-nodal) o Staging (I-IV)
o B symptoms
o Bulky disease >10 cm o Bone marrow involvement o FLIPI/IPI
o ECOG
- Treatment at diagnosis of FL and at transformation:
o Watch and wait
o Treatment (Chemotherapy, Immunotherapy, Radiotherapy, others …) o Allogeneic or autologous transplantation
Molecular and pathological data Project I
Fluorescence In Situ Hybridization (FISH)
Cytogenetic aberrations were analysed by FISH with the VysisCLL FISH Probe Kit, following the manufacturer's recommendations for detecting deletions of TP53 (17p13.1), ATM (11q22.3), D13S319 (13q14.3), MYC rearrangements/amplification (8q24.12-q24.13) and gain of the D12Z3 sequence (trisomy 12) in peripheral blood specimens from CLL patients.