ANALYSIS OF GENETIC CHANGES IN MYELOPROLIFERATIVE NEOPLASMS

20200129x_thesis_final_KubesovaDEPARTMENT OF INTERNAL MEDICINE – HEMATOLOGY AND ONCOLOGY
FACULTY HOSPITAL BRNO
Ph.D. thesis in the field of
Experimental Oncology and Tumor Biology
Supervisor: Author: Robert Kralovics, Ph.D. Mgr. Bc. Blanka Kubešová
Brno, 2020
Abstract
Introduction
Fully expanded TP53 mutations are rare in myeloproliferative neoplasms (MPN) but frequent
in post-MPN acute myeloid leukemia (AML). The multistep process of TP53 mutation
expansion during MPN transformation into AML has been documented retrospectively. Links
between TP53 mutations in post-MPN AML and cytoreduction with hydroxyurea (HU), a drug
triggering p53 response via replication stress, has been neither confirmed nor fully excluded.
Although MPNs harboring TP53 mutations with low variant allele frequency (VAF) were
described as “high risk”, it was unknown how common TP53 mutations were, whether they
were linked to HU cytoreduction and what disease progression risk they carried. This study
aimed to search for low-burden TP53-mutated subclones in chronic-phase-MPN patients and
to correlate presence of these clones with therapy, disease course, and other clinical features.
Methods
TP53 gene was investigated in MPN patients treated with HU, interferon α-2a or anagrelide and
in untreated patients using ultra-deep next generation sequencing (NGS) or functional yeast
analysis followed by NGS. In TP53-mutated cases, serial samples were analyzed.
Results
The analysis of 254 treated patients, as well as of 85 untreated patients, demonstrated that TP53
mutations occur irrespective of disease subtype, driver gene status and cytoreduction. TP53
mutations were found in 50 cases (0.2-16.3% VAF). Both therapy and TP53 mutations were
strongly associated with older age. In patients harboring TP53 mutations, retrospective and/or
prospective samples were examined if available to explore the clonal evolution of TP53-
mutated clones. Over-time analysis showed that the mutations may be undetectable at diagnosis
and slowly increase during disease course. Further, the impact of TP53 mutations on overall
survival and leukemic transformation was assessed. Although three patients with TP53
mutations progressed to TP53-mutated or TP53 wild-type AML, significant age-independent
impact on overall survival was not observed during the follow-up. Further, it was showed that
complete p53 inactivation alone led neither to blast transformation nor HU resistance.
Conclusion
Using highly sensitive method was showed that low-burden TP53 mutations are present in MPN
chronic phase. Patient`s age was revealed as the strongest factor affecting low-burden TP53
mutation incidence in MPN and no significant age-independent association between TP53
mutations and hydroxyurea were found. Monitoring of TP53 mutations during the disease
course showed that their clonal development is rather variable. Mutations may persist at low
levels for years without an immediate risk of progression. Nevertheless, TP53 minor mutations
may represent a pool for future clonal evolution.
Key words
Abstrakt
Úvod
Leukemická transformace je nepíliš astou, ale fatální, komplikací myeloproliferativních
neoplázií (MPN). Mutace v genu TP53 se v chronické fázi MPN objevují spíše vzácn, avšak
u sekundární akutní myeloidní leukémie (AML) je jejich výskyt astjší. Byly popsány pípady
expanze klonu s mutovaným genem TP53 v prbhu transformace. Souvislost vzniku nebo
selekce TP53 mutací a léby hydroxyureou (HU) nebyla potvrzena ani vyvrácena. Pestoe byla
pítomnost klonu s TP53 u MPN oznaena jako vysoce riziková, nebylo známo, jak asté
mutace genu TP53 v chronické fázi MPN jsou, jestli jsou asociovány s pedchozí terapií a jak
velké riziko progrese ve skutenosti nesou. Cílem této práce bylo zjistit, jestli a v jakém rozsahu
se mutace TP53 vyskytují v chronické fázi MPN, jestli jejich výskyt souvisí s pedchozí terapií
a jakou míru rizika pedstavují pro další vývoj onemocnní.
Metody
Gen TP53 byl u pacient s MPN vyšetován pomocí sekvenování nové generace (NGS) nebo
v pilotní analýze pomocí metody FASAY (Functional Analysis of Separated Alleles in Yeast)
a následn NGS. Byli vyšetování pacienti léení hydroxyureou, interferonem α-2a nebo
anagrelidem. V pípadech, kde byl nalezen mutovaný gen TP53, byly analyzovány následné
vzorky ke zjištní vývoje mutovaného klonu,
Výsledky
V rámci hlavní analýzy bylo vyšeteno 254 pacient léených hydroxyureou, interferonem α-
2a nebo anagrelidem a 85 pacient bez cytoredukní léby. Mutace v genu TP53 se u nich
vyskytovaly nezávisle na typu MPN, mutacích v “driver genech” a léb. Mutace genu TP53
byly celkov nalezeny u 50 pacient (0.2-16.3% VAF). Oba faktory, léba a výskyt TP53
mutací, byly siln asociovány s vyšším vkem pacient. Analýza následných vzork
odebraných v prbhu studie ukázala, e mutace mohou být v dob diagnózy nedetekovatelné
a jejich nálo me v ase narstat. Byl hodnocen také vliv pítomnosti mutací genu TP53 na
celkové peití a na progresi a transformaci do sAML. Nebyl pozorován ádný na vku
nezávislý vliv mutací TP53 na celkové peití. Ani kompletní inaktivace genu TP53 nevedla k
transformaci nebo k rezistenci na lébu hydroxyureou.
Závr
Analýza s vyuitím vysoce citlivých metod odhalila, e minoritní klony nesoucí TP53 mutace
jsou u pacient v chronické fázi MPN pítomny. Vk pacient se ukázal jako nejsilnjší faktor
ovlivující výskyt mutací TP53. ádná na vku nezávislá asociace mezi výskytem mutací TP53
a lébou hydroxyureou nalezena nebyla. Vývoj mutací v ase byl rznorodý. Minoritní
mutované klony mohou být pítomny dlouhodob bez akutního rizika progrese choroby, mohou
však slouit jako podhoubí pro budoucí klonální vývoj.
Klí ová slova
TP53, MPN, AML, transformace, hydroxyurea, interferon, anagrelid, NGS
I declare that I have carried out the work on this thesis independently under the
supervision of Robert Kralovics, Ph.D. and my consultant Mgr. Šárka Pavlová,
………………………………………………..
Acknowledgment
I would like to thank my supervisor Robert Kralovics, Ph.D. for help and for the opportunity
to spend 21 months working in the Research Centre for Molecular Medicine (CeMM) in
Vienna where I have met many inspiring people and got a lot of experience. I would like to
express my special thanks of gratitude to my consultant Mgr. Šárka Pavlová, Ph.D. for her
patience and huge help throughout the years. Without her valuable support, my study would
not have been completed. Furthermore, I would like to thank prof. RNDr. Šárka Pospíšilová,
Ph.D. and prof. MUDr. Michael Doubek. Ph.D. for their advice, RNDr. Jitka Malíková,
Ph.D. for help with manuscript, Mgr. Lenka Radová, Ph.D. for help with statistic analyses,
Mgr. Nikola Tom, Ph.D. for help with bioinformatic analyses, and all the hospitals involved
in samples’ collection for our study. Finally, I would like to express my gratitude to my
family for their never-ending support.
TABLE OF CONTENTS
1 INTRODUCTION ............................................................................................................. 10
1.2.1 Driver mutations – JAK2, CALR, MPL .............................................................. 16
1.2.2 Prognosis ............................................................................................................ 19
1.2.3 Non-driver mutations .......................................................................................... 20
1.2.4 Chromosomal aberrations ................................................................................... 25
1.2.5 Genetic aberrations associated with the process of disease progression ............ 26
1.3 Risk stratification ....................................................................................................... 31
1.4.1 Polycythemia vera .............................................................................................. 33
1.5.1 Hydroxyurea ....................................................................................................... 37
1.5.2 Anagrelide .......................................................................................................... 37
1.5.3 Interferon-α ......................................................................................................... 38
1.6.1 The role of TP53 in transformation .................................................................... 41
1.6.2 Association of previous treatment with disease progression and TP53 defects . 42
2 AIMS ................................................................................................................................. 44
3.2 Functional analysis of TP53 in yeast (FASAY) ........................................................ 45
3.3 TP53 Sanger sequencing ............................................................................................ 46
3.4 Colony-forming cell assay (CFC assay) .................................................................... 46
3.5 Ultra-deep next-generation sequencing of TP53 amplicons ...................................... 46
3.6 SNP arrays ................................................................................................................. 50
3.7 Statistical analysis ...................................................................................................... 50
4 RESULTS.......................................................................................................................... 51
4.1 Pilot study – minor TP53 mutations in chronic-phase MPN patients ........................ 51
4.2 Age-balanced study – low-burden TP53 mutations occur irrespective of hydroxyurea administration ....................................................................................................................... 56
4.3 Main analysis – ultra-deep NGS analysis of TP53 gene in treated MPN patients .... 58
4.3.1 Statistical analyses of the main cohort ............................................................... 64
4.3.2 Analysis of untreated patients ............................................................................ 74
4.3.3 Detailed description of TP53 mutations in HU-treated and other patients ........ 76
4.3.4 TP53 mutations may escape detection if examined at diagnosis........................ 79
4.3.5 Monitoring patients with TP53 mutations – dynamic behavior of mutated clones ………………………………………………………………………………….79
4.3.6 Impact of TP53 mutations on overall survival or leukemic transformation ....... 81
4.3.7 Summary of patients who died during the follow-up and developed sAML or clonal expansion ................................................................................................................ 84
4.3.8 Clonal expansion of TP53 mutated clone without transformation into sAML .. 87
5 DISCUSSION ................................................................................................................... 90
6 BIBLIOGRAPHY ............................................................................................................. 96
11 LIST OF PUBLICATIONS OF THE AUTHOR ........................................................ 123
11.1 First-author publication ............................................................................................ 123
11.2 Co-authored publications ......................................................................................... 124
11.3 Conference abstracts ................................................................................................ 125
13 ATTACHMENTS ....................................................................................................... 129
1.1 Myeloproliferative neoplasms
Myeloproliferative neoplasms (MPN) are a group of chronic diseases of bone marrow (BM)
characterized by a clonal hematopoiesis and an overproduction of differentiated fully functional
myeloid cells. The term myeloproliferative disorders (MPD) was first described by William
Dameshek in 1951 1. Later the description of recurrent mutations reclassified these diseases as
myeloproliferative neoplasms 2. The classification is multiparametric and consideres clinical
features, morphology and genetic data. The latest revision of the classification of myeloid
neoplasms from 2016 includes classical diseases as chronic myeloid leukemia (CML),
polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis
(prefibrotic/early PMF and overt fibrotic PMF). Further it includes chronic neutrophilic
leukemia, chronic eosinophylic leukemia not otherwise specified and MPN unclassifiable in
this group 3. A genetic change typical for CML is the reciprocal translocation between
chromosomes 9 and 22. This translocation results in a fusion gene BCR-ABL1 (Philadelphia
chromosome, Ph). The absence of this translocation is a characteristic feature of the other three
classical MPNs (Ph-negative) – PV, ET and PMF. These disorders share mutations that
constitutively activate the signal transduction pathway responsible for hematopoiesis 4. The
phenotype of one disease type can mimic phenotype of other disease types and even benign
hematopoietic disorders, such as hereditary thrombocytosis. This phenotype of benign
myeloproliferation can mask a clone of transformed hematopoietic stem cells, which could
develop and expand to acute myeloid leukemia.
1.1.1 Classification
The classification of myeloid neoplasms was revised in 2016 3. The reasons for the revision
consisted mainly of the expanded panorama of genetic lesions underlying MPNs and new
insights into the disease course derived from clinical studies. It is important to distinguish
individual MPN entities, because treatment strategies and survival differ. The World Health
Organization (WHO) classification for MPN from 2016 is described in Table 1.
11
Table 1: WHO classification of myeloid neoplasms and acute leukemia (adapted from WHO classification 2016 3).
12
Polycythemia vera is a chronic blood disorder characterized by erythrocytosis and often
leukocytosis and/or thrombocytosis. Further hallmarks of this disease are combinations of
splenomegaly, clonal hematopoiesis, endogenous erythroid colony growth and reduced serum
erythropoietin 5. The symptoms that patients are suffering from are hypertension, vascular
problems, headache, itching, night sweating, reddened face, weakness or dizziness. According
to revised World Health Organization (WHO) classification of myeloid neoplasms from 2016,
the first major criterion for PV diagnosis is increased hemoglobin or hematocrit or red cell
mass 3. The second major criterion is characteristic BM biopsy and the third criterion is
presence of mutation of JAK2 gene in exon 12 or 14 (V617F). The minor criterion is
subnormal serum erythropoietin level. Diagnosis of PV requires meeting all 3 major criteria,
or the first 2 major criteria and the minor criterion. The details about diagnosic criteria are
described in Table 2. The mutation of the gene JAK2 was described in 2005 and
pathophysiology of this condition was defined as JAK/STAT pathway activation, almost
always due to mutations in JAK2 exons 12 or 14 6–10. Incidence of PV ranges from 0.01 to
2.61 per 100,000 11. The most common complications are arterial and venous thrombosis due
to red-cell-mass induced hyperviscosity; transient ischemic attacks, ocular migraine, or
erythromelalgia due to activated platelets; aquegenic pruritus due to activated basophils;
acquired von Willebrand’s disease and pseudohyperkalemia due to extreme thrombocytosis;
splenomegaly due to extramedullary hematopoiesis. Occasionally, the disease may progress to
bone marrow failure, myelofibrosis or acute myeloid leukemia 4. Median survival of PV
patients is 10-16 years 11.
13
Table 2: WHO criteria for PV (adapted from WHO classification 2016 3).
1.1.3 Eseential thrombocythemia
Eseential thrombocythemia is characterized by thrombocytosis. It is the most indolent
myeloproliferative neoplasm 4. Patients are mainly asyptomatic. If present, the symptoms
include thrombosis or hemorrhage, headache, dizziness, chest pain, weakness, fainting,
temporary vision changes, numbness, tingling, or pain of the hands and feet, and less often
enlarged spleen. The major diagnostic criteria are platelet count higher than 450 x 109/l,
characteristic BM biopsy, not meeting WHO criteria for BCR-ABL1 positive CML, PV, PMF,
myelodisplastic syndromes, or other myeloid neoplasms, and the presence of JAK2, CALR, or
MPL mutation. The minor criterion is a presence of a clonal marker or an absence of the
evidence for reactive thrombocytosis. The diagnosis of ET requires meeting of all 4 major
criteria or the first 3 criteria and the minor criterion. The detailed description of diagnostic
criteria is showed in Table 3. The incidence of ET is estimated to 0.21-2.27 per 100,000 11.
Frequent complications of the disease are transient ischemic attacks, ocular migraine,
erythromelalgia, acquired von Willebrand’s disease and pseudohyperkalemia due to extreme
thrombocytosis; less common are arterial or venous thrombosis and transformation to bone
marrow failure, myelofibrosis or acute myeloid leukemia. This disease is a “diagnosis of
exclusion”, because isolated thrombocytosis may be present also in patients with polycythemia
14
vera or primary myelofibrosis. Median survival of ET patients is 10-22 years 12. Patients are
usually diagnosed at late middle age and have close to normal life expectancy 13.
Table 3: WHO criteria for ET (adapted from WHO classification 2016 3).
1.1.4 Primary myelofibrosis
Primary myelofibrosis is the least common and the most aggressive from classical MPNs 4. It
is manifested mainly as bone marrow fibrosis and splenomegaly due to extramedullary
hematopoiesis. There are more CD34+ cells circulating in peripheral blood, anemia, changes in
platelets and leukocyte counts, and constitutional symptoms due to inflammatory cytokine
production. In many patients, myelofibrosis is asymptomatic and is diagnosed by a routine
blood test or physical examination. In later stages general malaise, weight loss, or fever can
occur. There are two stages – prefibrotic/early primary myelofibrosis (prePMF) and overt PMF 3. The diagnostic criteria for prePMF and overt PMF are listed in Table 4. The diagnosis of both
disease strages requires meeting all 3 major criteria, and at least 1 minor criterion. It is necessary
to differentiate ET from prePMF, because this distinction has prognostic implications 3. The
incidence of PMF ranges from 0.22 to 0.99 per 100,000 11. The disease course is progressive,
characterized by BM failure, organ failure due to extramedullary hematopoiesis, and
transformation into acute myeloid leukemia in some cases. The presence of blasts in peripheral
blood (1-19%) defines an accelerated phase, whereas ≥20% blasts in peripheral blood is a
criterion for leukemia 14. PMF transforms more often to leukemia than PV and ET 15–17. Median
survival of PMF patients is 4-5 years 18.
15
Table 4: WHO criteria for prePMF and overtPMF (adapted from WHO classification 2016 3).
1.2 Genetic landscape and molecular pathophysiology of MPN
All MPN arise from a single somatically mutated hematopoietic stem cell (HSC) that clonally
expands 2. The expansion is accompanied by single or multilineage hyperplasia. PV is
characterized by erythroid lineage involvement as well as variable hyperplasia of
megakaryocytic and granulocytic lineages. ET and PMF are characterized by megykaryocytic
hyperplasia and PMF is defined by the presence of bone marrow fibrosis. Genetic landscape of
MPN has been mostly elucidated. There are several groups of mutations found in MPN patients
– mutations causing clonal hematopoiesis but not the typical phenotype of MPN, mutations
causing the MPN phenotype, and mutations leading to transformation of MPM to acute myeloid
leukemia (Figure 1). Mutations, which confirm the MPN diagnosis, are called driver mutations.
16
Figure 1: Model of disease initiation and progression in myeloproliferative neoplasms. Role of genetic lessions with different phenotypic effects (adapted from Milosevic et al 2013 19).
1.2.1 Driver mutations – JAK2, CALR, MPL
The most common mutations in MPNs are mutations in driver genes – Janus kinase 2 (JAK2),
calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL). These mutations
are implicated in the pathogenesis of MPN, required for the maintenance of the disease, and
essential for the myeloproliferative phenotype 2. All three mutations lead to the abnormal
ligand-independent activation of the cytokine receptor/Jak2 pathway and their downstream
effectors STATs which results in overproduction of myeloid cells (Figure 2).
17
1.2.1.1 Janus kinase 2 (JAK2)
As the first driver gene, mutation of JAK2 was described in 2005 6–9,20. JAK2 is located at
chromosome 9p24. The mutation is activating with a G to T somatic change at nucleotide 1849
resulting in the substitution of valine to phenylalanine at codon 617 in exon 14. JAK2 is a
member of JAK family of tyrosine kinases which includes also JAK1, JAK3, and TYK2 19,21.
The mechanism how the mutation in exon 617 causes the constitutive activation of the JAK2
kinase is based on the structure of the protein. It contains 4 funcional domains – FERM domain,
SH2 domain, pseudokinase domain (JH2), and tyrosine kinase domain (JH1). The pseudokinase
domain lacks kinase activity and its function is to negatively regulate the kinase domain and
the activity of the protein in general 22. The mutation V617F abrogates this function of the
pseudokinase domain. JAK2 helps to transduce signal from 3 main myeloid cytokine receptors
– erythropoietin receptor (EPOR), granulocyte colony-stimulating factor receptor (GCSFR),
and trombopoietin receptor (TPOR). After activation by ligand binding, these receptors induce
oligomerisation and phosphorylation of JAK2 proteins which is followed by further
phosphorylation of downstream targets 23. Small percentages of JAK2 V617F negative PV
patients carry mutations in exon 12 of the JAK2 gene 10,24. These mutations are located in the
linker between the SH2 domain and the pseudokinase domain and have similar functional
consequnces as mutated codon 617. JAK2 mutation is present in majority of PV patients (95 %)
and in 50-60 % of ET and PMF patients. In 2007, other mutations in JAK2 exon 12 were
described 10. These mutations are found mainly in myeloid lineages, but can be detected also in
lymphoid cells or even in endothelial cells 25. The heterozygous mutation at codon 617 can
undergo a transition to homozygous within a process of mitotic recombination which results in
a copy-neutral-loss of heterozygosity in the 9p region 6,26. The variant allele frequency (VAF)
can vary from the detection limit to 100 %. VAF is usually around 25 % in ET patients, over
50 % in PV patients and close to 100 % in post-PV and post-ET MF 27. JAK2 mutations were
found at wery low levels also in healthy population and even in neonate 28–30 and it is also
associated with clonal hematopoiesis of indeterminate potential common in elderly population 31. The order of mutations acquisition plays a role in several aspects. For example the order of
JAK2 and TET2 acquisition influences the phenotype of MPN disease 32. JAK2 is therapeutic
target and several JAK2 inhibitors were developed and more are in cilinical trials.
18
The development of megakaryocytes and platelets is regulated by thrombopoietin. After
binding to its receptor, it induces signaling through the JAK-STAT pathway. In 2006, MPL was
described as another driver gene 33. MPL is located at chromosome 1p34 and mutations were
described in exon 10 at the boundary of the transmembrane and the cytosolic domains. The
most frequent are mutations of tryptophan at codon 515. It is a gain-of-function substitution
leading to cytokine independent growth and thrombopoietin hypersensitivity in cell lines.
Mutations are usually heterozygous, but can become homozygous during the disease course 34.
MPL mutations were found in ET and PMF in 3% - 5% cases. 35,36. During the disease evolution,
MPL mutations arise rather early and can affect both myeloid and lymphoid lineages 37. Other
mutation of MPL, originaly described in hereditary form of thrombosis, is located in the
transmembrane domain, codon 505 38. Additionaly, non-canonical mutations of MPL were
described in triple negative MPN 39,40. These mutations cause amino acid changes in the
extracellular or intrcelular domains. They are more often acquired and lead to a true MPN.
Germline non-canonical MPL mutations can be also detected and suggest that…