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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS

CHRONIC LYMPHOCYTIC
LEUKEMIA
DIAGNOSIS, TREATMENT OPTIONS
AND PROGNOSIS

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CANCER ETIOLOGY, DIAGNOSIS
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CANCER ETIOLOGY, DIAGNOSIS AND TREATMENTS

CHRONIC LYMPHOCYTIC
LEUKEMIA
DIAGNOSIS, TREATMENT OPTIONS
AND PROGNOSIS

KIMBERLY RODRIQUEZ
EDITOR

New York
Copyright 2016 by Nova Science Publishers, Inc.

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CONTENTS

Preface vii
Chapter 1 The Chemokine Receptor CCR3 in Chronic
Lymphocitic Leukemia: Possible Biological Role
in the Course of the Disease 1
R. Vladimirova, E. Vikentieva, D. Popova, I. Gigov,
J. Raynov, A. Mihova and M. Guenova
Chapter 2 New and Repositioning Approaches to the Treatment
of Chronic Lymphocytic Leukemia 15
Ida Franiak-Pietryga and Maria Bryszewska
Chapter 3 Prognostic and Predictive Indicators in Chronic
Lymphocytic Leukemia 77
Nili Saar, Pia Raanani and Uri Rozovski
Chapter 4 Molecular Cytogenetic Abnormalities in Chronic
Lymphocytic Leukemia: Prognostic Implications 115
Prabhjot Kaur
Index 149
PREFACE

Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in


the western world, seen mostly in the elderly age-group and has a very
variable clinical outcome. Traditionally considered an indolent, antigen
inexperienced leukemia of slowly accumulating cells that do not die,
researchers now acknowledge that CLL cells are highly proliferative, antigen
experienced cells that have a high cell turnover and a subset show an
aggressive clinical course. The onset of the disease is usually asymptomatic;
only abnormalities in whole blood count such as leukocytosis with
lymphocytosis are found. Nowadays, CLL is diagnosed more often at an early,
asymptomatic stage due to more frequent routine blood tests. More advanced
stages are characterized by lymphadenopathy, hepatomegaly/splenomegaly,
recurrent infections, weakness, pallor and hemorrhagic diathesis, and general
symptoms such as weight loss, fever and night sweats are observed. This book
reviews the diagnosis, treatment options and prognosis of CLL.
Chapter 1 Chemokines and their receptors play a crucial role in cell
migration in physiological and pathological settings. The study focuses on the
impact of the expression of the chemokine receptor CCR3 in patients with
chronic lymphocytic leukemia (CLL). CCR3 is expressed upon a variety of
immune cells as well as on anaplastic large cell lymphoma cells. CCR3
expression on CLL-cells was reported in a small group of patients, though the
expression levels on B and T-cells and the clinical-laboratory correlations
remain unknown. Peripheral blood from a total of 91 untreated patients, staged
according to Rai Staging System and stratified into 3 risk groups, as well as 17
healthy controls were investigated.
The results showed that CCR3 expression on B-cells and T-cells was
significantly higher in CLL patients compared to the control group
viii Kimberly Rodriquez

(P < 0.001). CCR3 levels on B-cells didnt differ significantly among the risk
groups, while the expression on T-lymphocytes showed significant variations
(P = 0.020). A positive correlation between the levels of CCR3 on B-cells and
T-cells was found in the total group of patients, and particularly in stage Rai 0
(P < 0.001) and Rai I/II (P = 0.005). Cases with levels exceeding 10% of the
B-cells were characterized with higher WBC (P < 0.001), lymphocytes
(P = 0.003) and -cells absolute counts (P = 0.001). Positive correlations were
also found with -microglobulin (P = 0.027), Rai stage (P = 0.008) and risk
groups (P = 0.008). The higher the level of CCR3+ B-cells was detected, the
stronger the correlations were found.
These findings in regard to CCR3 in CLL-patients imply a possible
biological role of the receptor in the course of the disease. Besides, the high
levels of CCR3 on B-lymphocytes as well as on T-cells suggest a common
mechanism of activation of the two lymphocytic populations.
Chapter 2 Great progress has been made in the diagnosis and treatment
of Chronic Lymphocytic Leukemia (CLL), but this type of leukemia still
remains incurable, and the introduction of new drugs and new therapeutic
strategies are still desirable. For the last 20 years, significant progress in
molecular biology has resulted in better characterization and understanding of
the biology and prognosis of CLL. On the basis of recent research it has been
known that the critical role in the pathogenesis of CLL plays B-cell receptor
(BCR) activation and this provide new opportunities for the development of
innovative, more effective therapies. Recently, several small molecular kinase
inhibitors targeting the proximal BCR signaling pathway, including Brutons
tyrosine kinase (BTK) inhibitor (ibrutynib), and phosphatidylinositol 3-kinase
p110 (PI3Kp110) inhibitor (idelalisib), have been developed. These two
drugs are brand new, approved by US Food and Drug administration in 2014
and they are very promising in new personalized treatment strategy. Among
the new-targeted therapies, much attention is being paid to agents with a high
potential for triggering apoptosis in tumor cells. The PI3K/AKT signaling
pathway plays a crucial role in regulating survival of CLL B cells. The
inhibitors of this pathway, OSU-T315, MK2206 has also a strong antileukemic
activity in a very unique mechanism of action. Novel mechanism of abrogating
the AKT pathway and inhibition of BCR activity in CLL cells can be very
useful in the treatment of high risk leukemia, with del(17p13.1), unmutated
IGVH or those who may be resistant to ibrutinib.
Treatment approach in CLL has changed during last years. Purine
analogs (fludarabine, cladribine), pentostatins or other chemotherapeutics
(bendamustin) seem to be less effective and more toxic than monoclonal
Preface ix

antiobodies (rituximab, alemtuzumab). Preferable usage of these drugs is a


combination but the effectiveness leaves much to be desire. Why do not try
some other compounds? There are many research papers concerning surprising
and innovative approach to CLL treatment. Drug repositioning is an
application of known drugs and compounds to new indications (i.e., diseases).
A significant advantage of drug repositioning over traditional drug
development is that since the repositioned drug has already passed a
significant number of toxicity and other tests, its safety is known and the risk
of failure concerning adverse toxicology are reduced.
It had recently raised the meaning in CLL activity of known chemical
substances used for treatment of other disease such as valproic acid
(epilepsy) or atorvastatin (hypercholesterolemia), metformin (diabetes),
danazol (endometriosis). The new life for old drugs is very promising in CLL
treatment. The newest approach to CLL treatment focuses around
nanoparticles tested for example by our research team, leading us to
completely new and innovative kind of CLL treatment.
The aim of this article is to introduce the reader to the novelty in a current
and potential treatment options of CLL, as well as to open to new possibilities
and give chance to re-profiled substances - medication already existing on the
pharmaceutical market as adjuvants but in a brand new scene and with new
capabilities.
Chapter 3 Several prognostic factors are available for patients with CLL
who require therapy. Of these, high levels of 2 microglobulin, adverse
cytogenetics and high expression levels of CD38 and ZAP70 surface markers
are well described and mark patients with unfavorable prognosis. In
approximately 50% of patients, more than 2% of the immunoglobulin variable
heavy-chain gene (IGHV) nucleotide sequence differs from that of germ-line
non-clonal DNA. Patients with mutated IGHV respond better to chemo-
immunotherapy, and patients with unmutated IGHV have a shorter time to
treatment, shorter time to next treatment, inferior response to chemotherapy,
higher rates of chemotherapy resistance, and lower survival rates.
Until recently, CLL was considered an incurable disease. However,
two recent studies found that with the combination of fludarabine,
cyclophosphamide and rituximab (FCR) a fraction of patients with CLL
achieve long-term disease-free survival and are probably cured. Because none
of the novel agents that have been approved or are currently being tested cures
CLL, it has been argued that chemo-immunotherapy will remain the treatment
of choice, in particular for patients with mutated IGHV in the foreseen future.
x Kimberly Rodriquez

In this chapter the authors review the various prognostic markers that help
clinicians predict the clinical course and response to therapy of treatment nave
CLL patients. They highlight IGHV mutation status as a predictive marker that
could assist in selecting treatment from the growing arsenal of available drugs
for CLL.
Chapter 4 CLL is the most common leukemia in the western world.
Mostly a disease of the elderly, this indolent disorder, has a very variable
clinical outcome and can evolve into an aggressive lymphoma. As indolent
disorders are considered incurable, treatment is delayed until the patient
progresses to high stage disease. These therapeutic decisions are based on
criteria devised by Kanti Rai and Jacques Louis Binet almost four decades
ago; this assessment requires only a physical examination and a complete
blood count. A significant change that has occurred in the past four decades is
the increased diagnosis of patients in low stage disease (during regular check-
ups for employment or other causes). In the 1970s, 40% of the patients were
diagnosed at stage I. In the 2000s, nearly 80% of the patients are diagnosed at
low stage. Although patients are diagnosed at an earlier stage, it is difficult to
determine the prognosis of Rai stage 0/1 patients. A subset of patients with
early stage CLL will rapidly evolve to a more aggressive and fatal disease, and
clinical staging, as it currently exists, fails to predict this progression.
Approximately 80% of individuals with CLL have acquired chromosomal
abnormalities within their malignant clone and can be categorized into five
prognostic groups accordingly: deletion 13q (median survival, 133 months);
deletion 11q (median survival, 79 months); trisomy 12 (median survival, 114
months); normal cytogenetics (median survival, 111 months); and deletion 17p
(median survival, 32 months). A complex cytogenetic karyotype can be
identified rarely and is commonly associated with poor prognostic features
including CD38 expression and unmutated IgHV. IGHV3-21 usage is an
independent poor prognostic factor. Novel mutation in CLL include the
NOTCH1 mutations, SF3B1 mutations, BIRC3 mutation among others. B cell
receptors play a unique role in the pathogenesis of the disease and has
prognostic and therapeutic implications. Stereotyped B cell receptors impact
outcome. New hierarchal classification is being suggested to include the
conventional cytogenetic markers and novel mutations. MicroRNAs (miRNA)
are short (~22nt in humans), endogenous non-coding ssRNA (single stranded)
molecules that regulate gene expression via translational repression or
transcript degradation. Recent evidence has shown that miRNAs are key to
gene regulation and have a global effect on various oncogenic, tumor
suppressor and cell survival pathways. Over the last decade several miRNAs
Preface xi

have been shown to be involved in the pathogenesis of CLL. In this chapter,


molecular cytogenetic prognostic markers, BCRs, novel mutations, role of
miRNA in the biology and prognosis of this disease are discussed.
In: Chronic Lymphocytic Leukemia ISBN: 978-1-63485-510-5
Editor: Kimberly Rodriquez 2016 Nova Science Publishers, Inc.

Chapter 1

THE CHEMOKINE RECEPTOR CCR3


IN CHRONIC LYMPHOCITIC LEUKEMIA:
POSSIBLE BIOLOGICAL ROLE IN
THE COURSE OF THE DISEASE

R. Vladimirova1,*, E. Vikentieva1, D. Popova1,


I. Gigov1, J. Raynov1, A. Mihova2 and M. Guenova2
1
Military Medical Academy, Sofia, Bulgaria
2
National Specialised Hospital for Active Treatment of Hematological
Diseases, Sofia, Bulgaria

ABSTRACT
Chemokines and their receptors play a crucial role in cell migration
in physiological and pathological settings. The study focuses on the
impact of the expression of the chemokine receptor CCR3 in patients
with chronic lymphocytic leukemia (CLL). CCR3 is expressed upon a
variety of immune cells as well as on anaplastic large cell lymphoma
cells. CCR3 expression on CLL-cells was reported in a small group of
patients, though the expression levels on B and T-cells and the clinical-
laboratory correlations remain unknown. Peripheral blood from a total of
91 untreated patients, staged according to Rai Staging System and
*
Corresponding author: Rositsa Vladimirova Andreeva, Department Cytogenetic and
immunology Military Medical Academy, Bulgaria 1606 Sofia 3 St. Georgi Sofiiski
Street, Tel: +359 888 351 777, e-mail: rossy_vladimirova@yahoo.com.
2 R. Vladimirova, E. Vikentieva, D. Popova et al.

stratified into 3 risk groups, as well as 17 healthy controls were


investigated.
The results showed that CCR3 expression on B-cells and T-cells was
significantly higher in CLL patients compared to the control group
(P < 0.001). CCR3 levels on B-cells didn't differ significantly among the
risk groups, while the expression on T-lymphocytes showed significant
variations (P = 0.020). A positive correlation between the levels of CCR3
on B-cells and T-cells was found in the total group of patients, and
particularly in stage Rai 0 (P < 0.001) and Rai I/II (P = 0.005). Cases
with levels exceeding 10% of the B-cells were characterized with higher
WBC (P < 0.001), lymphocytes (P = 0.003) and -cells absolute counts
(P = 0.001). Positive correlations were also found with -microglobulin
(P = 0.027), Rai stage (P = 0.008) and risk groups (P = 0.008). The
higher the level of CCR3+ B-cells was detected, the stronger the
correlations were found.
These findings in regard to CCR3 in CLL-patients imply a possible
biological role of the receptor in the course of the disease. Besides, the
high levels of CCR3 on B-lymphocytes as well as on T-cells suggest a
common mechanism of activation of the two lymphocytic populations.

Keywords: chronic lymphocytic leukemia, CCR3, B-lymphocytes,


T-lymphocytes

INTRODUCTION
Chronic lymphocytic leukemia (CLL) is a neoplasm of B-lymphocytes
immunophenotypically characterized by the aberrant coexpression of CD5,
CD23 and forming proliferation centers in tissue infiltrates [1]. Although
generally with indolent clinical course, the disease poses serious medical,
pharmaco-economic and social problems in the clinical practice. CLL is a
heterogeneous disease at the clinical, molecular and cellular levels and
survival times vary widely depending on the clinical stage and risk group [2].
In recent years, intensive studies have been aimed at clarifying the main
pathogenetic mechanisms and therapeutic options. Chemokines and the
corresponding receptors play a crucial role in tumor progression and tumor
cell dissemination [3]. Up to now, several chemokines have been demonstrated
to be involved in the migration of CLL cells to lymph nodes, secondary
lymphoid organs and bone marrow. Besides, some chemokines are known to
have an anti-apoptotic effect and thus contribute to the survival of CLL cells
[4].
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 3

The chemokine receptor CCR3 (CD193) is expressed upon a variety of


immune cells, including eosinophils [5, 6], Th2-lymphocytes [7], mast cells
[8], and basophils [9, 10]. It has been shown that in Hodgkin lymphoma and
cutaneous T-cell lymphoma, cancer associated fibroblasts secrete the
chemokines CCL11 and CCL26 (CCR3 ligands) that recruit CCR3+
T-lymphocytes into the tumor and produce high levels of IL-4, a signature of a
Th2-dominant microenvironment [12, 13]. Besides, CCR3 is a specific marker
of anaplastic large cell lymphoma cells, where the CCR3 - CCL11 interaction
promotes tumor cell proliferation and inhibits apoptosis through ERK1/2, Bcl-
xL and the production of survivin [13, 14].
Progressive CLL is characterised by the accumulation of neoplastic B-
cells in the tissues, moreover in the specific microenvironment monoclonal B-
cells interact with T-cells. The CCR3 molecule has not been found on normal
B-lymphocytes, however one study investigated the role of its expression on
malignant B-lymphocytes including 13 patients with CLL. CCR3 was found in
almost all CLL patients as the positive neoplastic B-cells showed increased
migratory activity [11].
Therefore we aimed our study at investigating the impact of the
chemokine receptor CCR3 expression on B- and T-lymphocytes in CLL
patients in correlation with major clinical and laboratory data in order to reveal
a possible role for the biological course of the disease.

MATERIALS AND METHODS


Patients

A total of 91 previously untreated patients with CLL (49 men and 42


women) diagnosed in Department of Haematology of Military Medical
Academy and the National Specialised Hospital for Active Treatment of
Hematological Diseases, Bulgaria were enrolled in the study. The median age
of patients was 67 years (range: 3784 years). The patients were staged
according to Rai Staging System and stratified into 3 risk groups: low risk (Rai
0), intermediate (Rai I/II) and high risk (Rai III/IV) (Table 1). The control
group of 17 age-matched healthy persons was also included, 11 male and 6
female at a median age of 63 years (range: 3978 years). The study was
approved by the local ethics committee and informed consent was obtained
from all subjects prior to enrolment in accordance with the Declaration of
4 R. Vladimirova, E. Vikentieva, D. Popova et al.

Helsinki. Diagnosis was based on the criteria of WHO Classification of


Tumours of Haematopoietic and Lymphoid Tissues, Fourth Edition [1].

Flow Cytometry

Immunophenotyping was performed on lysed and washed peripheral


blood in 2EDTA diluted to approximately 1 x 106/ml with FACS Wash
Buffer (BD Biosciences, San Jose, CA, USA). The investigated antigens were
detected by a direct immunofluorescence assay using a panel of monoclonal
antibodies (Moabs) according to procedures prescribed by the manufacturer.
For each tube 50 l from the diluted sample were incubated with 5 l of each
fluorochrome-conjugated monoclonal antibody for 30 minutes in the dark at
room temperature (20 to 25C), followed by 10 minutes lysing with 2 ml
FACS Lysing Solution (BD Biosciences, San Jose, CA, USA) and washing to
remove excess antibody and debris. Permeabilization procedure by 2 hours
incubation with 2 ml FACS Lysing Solution was performed for Zap-70 and
CD79a immunolabeling. Cell samples were analyzed by six-colour flow
cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA) using the
FACSDiva 6.0 software (BD). At least 50000 events were acquired. The
phenotyping panel of Moabs included: CD19, CD20, CD22, CD79a, CD79b,
CD5, CD23, CD3, CD4, CD8, CD16+56, Zap-70, CD38, CD49d, and
light chains (BD Biosciences, San Jose, , USA). CCR3 was detected by PE
Mouse Anti-Human CD193; Clone: 5E8 (BD Pharmingen). The flow
cytometry results were presented as follows: CD19+ cells were presented as
percentage out of lymphocytes, as well as an absolute count; in regard to other
antigens the percentage of positive cells out of CD19+ B-cells was estimated,
while CCR3 expression was analyzed also on CD3+ T-cells; the percentage of
Zap-70+ out of the CD79a+ population was included in the analysis.

Table 1. Distribution of patients by sex, age, Rai stage and risk groups

Male (%) Female (%)


Sex 49 (53.85) 42 (46.15)
Distribution by age 37 45 46 55 56 65 66 75 76 84
Patients count (%) 4 (4.39) 9 (9.89) 28 (30.81) 33 (36.3) 17 (18.7)
Stage to Rai staging Rai 0 Rai I Rai II Rai III Rai IV
system Patients count (%) 30 (32.97) 17 (18.68) 16 (17.58) 14 (15.38) 14 (15.38)
Distribution by risk Rai 0 Rai I/II Rai III/IV
Patients count (%) 30 (32.97) 33 (36.26) 28 (30.77)
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 5

Statistical Analysis

Statistical analysis was carried out using the SPSS 21.0 software package
(SPSS Inc., Chicago, Illinois, USA). Continuous data parameters were
analyzed for normality using the W-ShapiroWilk test; data were presented as
median and (P10 P90), because variables had nonparametric distribution. A
nonparametric U-MannWhitney test and KruskalWallis test (for continuous
variables) were used. Spearmans rank correlation () was used to measure the
relationship between variables. Groups were assumed to differ significantly
when the P value was less than 0.05 and highly significant when the P value
was less than 0.001.

RESULTS
Flow cytometry allowed for detecting CCR3 both on normal and leukemic
B-cells as well as on T-cells in all samples of enrolled patients and healthy
controls though in a very wide range: 0.2 - 100% of B-cells and 0.1 - 100% of
T-cells. The percentage of positive B-cells was significantly higher in
leukemic samples compared with healthy controls median value and P10 -
P90 was 5% (0.7 40.6) vs. 0.5% (0.2 2.0), respectively (P < 0.001). The
same was found in regard to T-cells: the median value and P10 - P90 was
22.4% (1.5 98.0) in CLL vs. 1.4% (0.1 - 17.7) in the healthy control group
(P < 0.001) (Table 2). In addition, the highest levels of CCR3 expression on
B-cells as well as on T-cells were detected in high-risk patients, however,
being statically significant only in regard to the T-cell compartment
(P = 0.020) (Table 2).
Chemokine receptor CCR3 expressed by monoclonal B-lymphocytes
show positive correlation with the expression of CCR3 on T-cells in the total
group of patients with CLL (P < 0.001). The expression of CCR3 on T-
lymphocytes shows sufficient positive correlation with the expression levels of
CD49d (P = 0.006) (Table 3). Expression of CCR3 higher than 10% was
found in 28 patients throughout all risk groups. There were 10 cases in the low
risk group, 6 cases in the intermediate risk group (Rai I 4; Rai II 2) and
12 patients in the high-risk group (Rai III 5; Rai IV - 7). The CCR3 values in
these patients had positive correlations to the absolute count of leukocytes (P <
0.001), lymphocytes (P = 0.003), monoclonal B-cells (P = 0.001), to the
percentages of lymphocytes (P = 0.047) and monoclonal lymphocytes (P =
0.028), as well as to the 2-microglobulin concentration (P = 0.027). The
6 R. Vladimirova, E. Vikentieva, D. Popova et al.

positive correlations with the Rai stage (P = 0.008) and risk groups (P =
0.008) are moderate. The correlation between CCR3 and the haemoglobin
level was negative (P = 0.017) (Table 3). Expression of CCR3 higher than
20% was found in 18 patients, with 61% of them belonging to the high risk
group, 4 patients were in the low risk group, 3 patients in the intermediate
risk group (Rai I 1; Rai II 2) and 11 patients in the high risk group (Rai III
5 Rai IV - 6), in other words 7 patients were in the low and intermediate risk
groups, and 11 patients belonging to the high risk group, with 56 of the rest of
the patients being in the low and intermediate risk groups and 17 patients in
the high-risk group. In the case of this higher level of expression of the
receptor (%CCR3+ B-cells > 20%), the existing correlations with absolute
count of leukocytes, lymphocytes, monoclonal B-cells, with the percentage of
lymphocytes and B-lymphocytes as well as with 2-m concentration increase
their power (Table 3).
The correlations of the studied chemokine receptor changed within the
risk groups. The correlation between the expression of chemokine receptor on
the B- and T-cells is the strongest in the low risk group (P < 0.001), the
intensity of this correlation decreased in the intermediate risk group (P =
0.005) and was not found in high-risk patients. Aberrant expression of CCR3+
on monoclonal B-lymphocytes in the Rai 0 stage shows negative correlation to
platelets count (P = 0.022) and positive correlation to the -cells percentage
(P = 0.019) and the expression of Zap-70 (P = 0.010), while within the high-
risk group only the positive correlation to the expression of CD49d (P = 0.022)
was observed (Table 4). Inflammatory chemokine receptor CCR3 on T-
lymphocytes shown positive correlation with the levels of CD49d expressing
B-cells in the intermediate risk group (P = 0.014) (Table 4).

Table 2. CCR3 expression on B and T-lymphocytes

Rai Rai
Control CLL P Rai 0 P
I/II III/IV
(n = 17) (n = 91) value (n = 30) value
(n = 33) (n = 28)
%
0.5 5.0 5.6 3.7 7.7
CCR3+ <0.001 NS
(0.2 - 2.0) (0.7 - 40.6) (0.7 - 21.8) (1.0 - 22.2) (0.6 - 72.8)
B-cells
%
1.4 22.4 20.8 5.5 53.4
CCR3+ <0.001 0.020
(0.1 - 17.7) (1.5 - 98.0) (0.6 - 90.8) (0.62 - 90.8) (3.41 - 99.7)
T-cells
Median and (10 90); NS - not significant
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 7

Table 3. The correlation between laboratory parameters and the


expression of receptors CCR3 on B and T-cells

% CCR3+ % CCR3+ B-cells > 10% % CCR3+ T-cells


Patients with CLL B-cells
n = 91 n = 28 n = 91
WBC [x109/l] NS P < 0.001 ( = + 0.608) NS
*P < 0.001 ( = + 0.777)
Ly [%] NS P = 0.047 ( = + 0.378) NS
*P = 0.009 ( = + 0.600)
Ly [x109/l] NS P = 0.003 ( = + 0.548) NS
*P < 0.001 ( = + 0.750)
CD19+ [%] NS P = 0.028 ( = + 0.415) NS
*P = 0.020 ( = + 0.543)
CD19+ [x109/l] NS P = 0.001 ( = + 0.613) NS
*P < 0.001 ( = + 0.754)
HGB x [g/dl] NS P = 0.017 ( = - 0.447) NS
PLT [x109/l] NS NS NS
LDH [U/l] NS NS NS
2-m [mg/l] NS P = 0.027 ( = + 0.433) NS
*P = 0.044 ( = + 0.627)
CD38+ B-cells [%] NS NS NS
CD49d+ B-cells [%] NS NS P = 0.006 ( = + 0.285)
Zap-70+ B-cells [%] NS NS NS
Rai stage NS P = 0.008 ( = + 0.508) NS
Risk groups NS P = 0.008 ( = + 0.509) NS
CCR3+ B-cells [%] - - P < 0.001 ( = + 0.531)
* % CCR3+ B-cells > 20%: n = 18; NS - not significant

Table 4. The correlation between laboratory parameters and the


expression of the receptor CCR3 on B and T-cells in groups by risk

Parameter Rai 0 Rai I/II Rai III/IV


CCR3+ B-cells [%]
P = 0.022 NS NS
PLT [x109/l]
( - 0.416)
P = 0.019 NS NS
CD19+ [%]
( + 0.427)
P = 0.010 NS NS
Zap-70+ B-cells [%]
( + 0.473)
NS NS P = 0.022
CD49d+ B-cells [%]
( + 0.447)
P < 0.001 P = 0.005 NS
CD193+ T-cells [%]
( + 0.716) ( + 0.478)
CCR+ T-cells [%]
NS P = 0.014 NS
CD49d+ B-cells [%]
( + 0.425)
8 R. Vladimirova, E. Vikentieva, D. Popova et al.

DISCUSSION
Chemokines and their receptors play an important role in the tumour
development process, by directing cell traffic to the tumour microenvironment
or facilitating its exit from it. The general conclusion from the scientific
literature data brings to the fore the detection of aberrantly expressed
chemokine receptors from tumour cells [15].
Chemotaxic receptor CCR3 connects with a wide range of ligands -
CCL5, CCL2, CCL7, CCL11 (eotaxin), CCL13, CCL15, CCL24 (eotaxin - 2),
CCL26 (eotaxin - 3) and CCL28 with all bonds causing activation of the
receptor. The ligands of CCR3 bind it through a variable-strength connection,
with it being the strongest for CCL11, followed by CCL13 > CCL26 > CCL24
and CCL5 [10]. The binding of this eotaxin activates intracellular signals, the
final result being activation and migration.
A study of CCR3 expression in a small number of patients with CLL
found aberrant expression of the receptor on neoplastic cells and establishes
the higher migration activity of B-lymphocytes expressing CCR3 [11].
Therefore we aimed at investigating the chemokine receptor CCR3 (CD193)
expression on B- and T-lymphocytes in a larger cohort of CLL patients in
comparison to healthy controls and to correlate data with major clinical and
laboratory data in order to reveal a possible role for the biological course of
the disease.
In the present study we found increased levels of aberrant expression on
B-cells of patients with CLL compared to healthy controls, with the highest
levels of CCR3 reported in the high-risk group, as well as significantly higher
levels of positive T-lymphocytes. The proportion of positive monoclonal B-
cells showed a significant correlation with the CCR3-positive T-cells in CLL
patients which could suggest the presence of a common mechanism of
activation of the B- and T-cells through CCR3 in the case of disease. Cytokine
or chemokine stimulation in patients with CLL could probably be the reason
for the presence and the degree of this aberrant expression as there is at least
one in vitro study of isolated peripheral-blood and germinative B-cells that
proved the ability of B-lymphocytes to express CCR3 after stimulation with
IL-2 and IL-4 [16].
With the increase of CCR3 levels on B-lymphocytes an increasingly
strong connection between the receptor and adverse clinical factors such as
higher tumour burden, translated into higher leukocyte, lymphocyte and
monoclonal B-cell counts, 2-microglobulin levels, as well as decreased
haemoglobin levels and higher Rai stages, respectively could be found.
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 9

Based on these observations, the importance of CCR3 aberrant expression


in leukemogenesis and clinical behaviour, respectively, could be speculated
and discussed in the light of other studies on CCR3 ligands in CLL. Data are
available that the connection between CCR3 and the eotaxin, the ligand with
the strongest binding potential, has not only a migrational but also angiogenic
activity [17]. The eotaxin is present in higher concentrations in the serum of
patients with CLL [18], and the natural antagonist of this connection CXCR3
[19] is expressed in lower levels on the leukemic cell [20]. Therefore, it can be
assumed that the receptor-ligands levels play an important role in the
disease. Besides, the changes in the correlation dependencies between CCR3
expression on B- and T-lymphocytes and the adhesion and activation
molecules in the risk groups probably reflect the different microenvironment
stimuli on the lymphocytes and the disease dynamics.

CONCLUSION
The findings from the present study of the increased expression of CCR3
in patients with CLL and correlations with the disease characteristics clearly
demonstrate the role of this aberrant expression in the biological course of
disease. Further studies are warranted in order to elucidate the precise
mechanisms of possible common activation of the B- and T-cell compartments
and the pathogenetic consequences which might be the basis for investigating
novel therapeutic approaches.

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Okochi, H; Tamaki, K; Sato, S. CCL11-CCR3 interactions promote
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[20] Ocana, E; Delgado-Perez, L; Campos-Caro, A; Muz, J; Paz, A;
Franco, R; Brieva, JA. The prognostic role of CXCR3 expression by
chronic lymphocytic leukemia B cells. Haematologica, 2007, 92, 349
56.

BIOGRAPHICAL SKETCH
Name: Rositsa Vladimirova Andreeva
Affiliation: Member, European Research Initiative on CLL
Member, Bulgarian Medical Society of Hematology
Member, Bulgarian Association for Clinical Immunology
Education: 1990 Master of Biology - Sofia University St Kliment
Ohridski Faculty of Biology
2014 PhD Military Medical Academy, Sofia; Bulgaria; Hematology
Address: Bulgaria; 1606 Sofia,
Department Cytogenetic and Immunology
Military Medical Academy
3 St. Georgi Sofiiski Street
Research and Professional Experience: Flow cytometry in neoplastic
hematology; Clinical Immunology
12 R. Vladimirova, E. Vikentieva, D. Popova et al.

Professional Appointments: Assistant Professor, Department of


Cytogenetic and Immunology
Publications:

1. Vladimirova R, Popova D, Vikentieva E, Guenova M. Chronic


Lymphocytic Leukemia: Microenvironment and B Cells. Leukemias -
Updates and New Insights; Prof. Margarita Guenova (Ed.), ISBN:
978-953-51-2202-9, In Tech, DOI: 10.5772/60761.
2. Vladimirova R, Vikentieva E, Popova D, Kindekov I, Nikolov I,
Stanchev R, Petkova N, Gigov I, Raynov J. Phenotypic Profiles in
Chronic Lymphocytic Leukemia Prerequisite for Different Clinical
Course. Medical Review 2016; In press.
3. Vladimirova R, Vikentieva E, Popova D, Kindekov I, Nikolov I,
Stanchev R, Damianov I, Kancheva T, Petkova N, ilcheva K,
Nedeva A, Gigov I, ihova A, Guenova M, Raynov J. Chronic
Lymphocytic Leukemia: CD184 Expression and Correlations.
Hematology 2016; in press.
4. Kyosev, Kazakova M, Popova D, Vladimirova R, Vikentieva E,
Mutafchiiski, Vasilev K, Serafian V. Colorectal Cancer Surgical
Approaches and YKL-40 Serum Levels. Endourology and Minimally
Invasiva Surgery 2015; 3. 1: 38-44.
5. R. Vladimirova, D. Popova, E. Vikentieva, I. Nikolov, R. Stanchev, I.
Damianov, T. Kuncheva, N. Petkova, K. Milcheva, I. Kindrkov, I.
Gigov, J. Rainov. Cell Adhesion Molecules Expression in Chronic
Lymphocytic Leukemia. Hematology 2015; LI. 1-2: 58-63.
6. Vladimirova R, Popova D, Vikentieva E. Chronic Lymphocytic
Leukemia Clinical and Biological Prognostic Factors. Bulgarian
Association for Clinical Immunology 2014; 7: 35-47.
7. Vladimirova R, Popova D, Vikentieva E, Galabova I, Mitev L, Gigov
I, Rainov J. Expression on Costimulatory Receptors in Chronic
Lymphocytic Leukemia Relationship with Progression of Disease
Medical Review 2014; L. 3: 31-36.
8. Kazakova M, Vladimirova R, Vikentieva E, Popova D, Serafian V.
YKL-40 A potential prognostic factor in chronic lymphocytic
leukemia? Science & Technologies, 2014; 4. 1: 277-281.
9. Mitev L., Velizarova M., Uzunova V., Stanchev R., Gigov I., Rainov
J., Vladimirova R., Tsachev K. Three Cases of Acute Myeloid
Leukemia and Deletion of The Long Arm of Chromosome 9. Acta
Medica Bulgarica 2013; XL.1:10-17.
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 13

10. Vladimirova R, Popova D, Vikentieva E, Girova N, Ramsheva Z,


Polomski N, Mitev L. Prognostic Levels of Serum Immunoglobulins
in Chronic Lymphocytic Leukemia Bulgarian Association for Clinical
Immunology 2013; 6: 40-49.
11. Vladimirova R, Popova D, Vikentieva E, Mitev L. Chronic
Lymphocytic Leukemia Chemokine Network Migration,
Antiapoptotic and Activation Signals. Clinical and Transfusion
Hematology 2013; XLVIV. 1-2: 9-16.
12. Vladimirova R, Vikentieva E, Popova D, Mitev L, Nikolov I,
Stanchev R, Damianov I, Kuncheva T, Petkova N, Milcheva K, Gigov
I, Rainov J. Migration and Antiapoptotic Markers as Potencial
Predictors in Chronic Lymphocytic Leukemia. Clinical and
Transfusion Hematology 2013; XLVIV. 1-2: 60-63.
13. Vladimirova R, Galabova I, Vikentieva E, Popova D, Mitev L,
Nikolov I, Stanchev R, Damianov I, Kuncheva T, Petkova N,
Milcheva K, Gigov I, Rainov J. CD80/CD86 Expression in Chronic
Lymphocytic Leukemia Relationship with Proliferation Activity.
Clinical and Transfusion Hematology 2013; XLVIV. 1-2: 64 - 68.
14. Vladimirova R, Vikentieva E, Nikolov I, Stanchev R, Mitev L,
Popova D. Chronic Lymphocytic Leukemia v/s Persistent Polyclonal
B-cell Lymphocytosis Clinical and Transfusion Hematology 2013;
XLVIV. 1-2: 69 - 72.
15. Vladimirova R, Popova D, Vikentieva E, Mitev L. Mature B-cell
Lymphoproliferative Neoplasms Common Cytogenetic Anomalies.
Bulgarian Association for Clinical Immunology 2012; 5: 36-54.
In: Chronic Lymphocytic Leukemia ISBN: 978-1-63485-510-5
Editor: Kimberly Rodriquez 2016 Nova Science Publishers, Inc.

Chapter 2

NEW AND REPOSITIONING APPROACHES TO


THE TREATMENT OF CHRONIC
LYMPHOCYTIC LEUKEMIA

Ida Franiak-Pietryga1,* and Maria Bryszewska2


1
Department of Clinical and Laboratory Genetics,
Medical University of Lodz, Poland
Department of General Biophysics, Faculty of Biology
and Environmental Protection, University of Lodz, Poland

ABSTRACT
Great progress has been made in the diagnosis and treatment of
Chronic Lymphocytic Leukemia (CLL), but this type of leukemia still
remains incurable, and the introduction of new drugs and new therapeutic
strategies are still desirable. For the last 20 years, significant progress
in molecular biology has resulted in better characterization and
understanding of the biology and prognosis of CLL. On the basis of
recent research it has been known that the critical role in the pathogenesis
of CLL plays B-cell receptor (BCR) activation and this provide new
opportunities for the development of innovative, more effective therapies.
Recently, several small molecular kinase inhibitors targeting the
proximal BCR signaling pathway, including Brutons tyrosine kinase

*
Corresponding Ida Franiak-Pietryga, Department of Clinical and Laboratory Genetics, Medical
University of Lodz, Pomorska 251, 92-213 Lodz, Poland; Telephone/Fax: +48 42 272 53
60; Email: ida.fp@interia.pl; ida.franiak-pietryga@umed.lodz.pl.
16 Ida Franiak-Pietryga and Maria Bryszewska

(BTK) inhibitor (ibrutynib), and phosphatidylinositol 3-kinase p110


(PI3Kp110) inhibitor (idelalisib), have been developed. These two drugs
are brand new, approved by US Food and Drug administration in 2014
and they are very promising in new personalized treatment strategy.
Among the new-targeted therapies, much attention is being paid to agents
with a high potential for triggering apoptosis in tumor cells. The
PI3K/AKT signaling pathway plays a crucial role in regulating survival
of CLL B cells. The inhibitors of this pathway, OSU-T315, MK2206 has
also a strong antileukemic activity in a very unique mechanism of action.
Novel mechanism of abrogating the AKT pathway and inhibition of BCR
activity in CLL cells can be very useful in the treatment of high risk
leukemia, with del(17p13.1), unmutated IGVH or those who may be
resistant to ibrutinib.
Treatment approach in CLL has changed during last years. Purine
analogs (fludarabine, cladribine), pentostatins or other chemotherapeutics
(bendamustin) seem to be less effective and more toxic than monoclonal
antiobodies (rituximab, alemtuzumab). Preferable usage of these drugs is
a combination but the effectiveness leaves much to be desire. Why do not
try some other compounds? There are many research papers concerning
surprising and innovative approach to CLL treatment. Drug repositioning
is an application of known drugs and compounds to new indications (i.e.,
diseases). A significant advantage of drug repositioning over traditional
drug development is that since the repositioned drug has already passed a
significant number of toxicity and other tests, its safety is known and the
risk of failure concerning adverse toxicology are reduced.
It had recently raised the meaning in CLL activity of
known chemical substances used for treatment of other disease
such as valproic acid (epilepsy) or atorvastatin (hypercholesterolemia),
metformin (diabetes), danazol (endometriosis). The new life for old drugs
is very promising in CLL treatment. The newest approach to CLL
treatment focuses around nanoparticles tested for example by our
research team, leading us to completely new and innovative kind of CLL
treatment.
The aim of this article is to introduce the reader to the novelty in a
current and potential treatment options of CLL, as well as to open to new
possibilities and give chance to re-profiled substances - medication
already existing on the pharmaceutical market as adjuvants but in a brand
new scene and with new capabilities.

Keywords: chronic lymphocytic leukemia, new treatment, repositioning


treatment, nanoparticles
New and Repositioning Approaches to the Treatment 17

1. INTRODUCTION
Chronic lymphocytic leukemia (CLL) is a type of leukemia most
commonly diagnosed in Western Europe and North America, with an
incidence rate of 4.2/100,000 [1] Although the median age at initial diagnosis
is 67-72 years [2], with approximately 70% of patients being over 65, and a
male to female ratio of 1.7:1 [3], CLL is now increasingly affecting younger
patients between 20 and 60 years old. The only confirmed risk factor is a
family history of this disease. For relatives of CLL patients, the risk increases
by a factor of 2.5-7.5 [4].
The onset of the disease is usually asymptomatic; only abnormalities in
whole blood count such as leukocytosis with lymphocytosis are found.
Nowadays, CLL is diagnosed more often at an early, asymptomatic stage due
to more frequent routine blood tests. More advanced stages are characterized
by lymphadenopathy, hepatomegaly/splenomegaly, recurrent infections,
weakness, pallor and hemorrhagic diathesis, and general symptoms such as
weight loss, fever and night sweats are observed.
In recent years, there has been significant progress in the treatment
of CLL, firstly thanks to the introduction of immunochemotherapy with
monoclonal antibodies, and also through the use of small molecules, such as
tyrosine kinase inhibitors, targeting B-cell receptor signaling. Alkylating
agents and glucocorticoids were first used in the treatment of CLL in the
1950s [5, 6]. Purine analogues (cladribine, fludarabine and pentastatin) were
introduced in the 1980s. The development of rituximab by IDEC
pharmaceuticals and its subsequent FDA approval for treatment of non-
Hodgkin lymphoma in 1997 introduced chemoimmunotherapy regimens
which to this day remain the standard approach to initial therapy of younger
patients with CLL [7]. Interestingly, prednisone was the first agent that could
be regarded as an example of targeted therapy in CLL. Steroid hormones
modulate many intracellular pathways, two of which allow it to target
lymphoid malignancies. Firstly, glucocorticoid receptors interfere with the
activity of the nuclear factor B (NF-B) pathway; by inducing the
production of the inhibitory protein inhibitor B (IB), they promote
sequestration of NF-B in the cytoplasm [8]. This impairs transcription of NF-
B target genes and reduces cell proliferation and survival. Secondly,
glucocorticoids bind and inhibit AP-1 transcription factor activity, which is
necessary for cell proliferation [9].
Frontline therapies for CLL have been based on the administration of
cytostatic drugs (chlorambucil, fludarabine), which in many cases, control the
18 Ida Franiak-Pietryga and Maria Bryszewska

disease efficiently and are well tolerated [2]. However, patients carrying
certain prognostic markers, such as del(17p) or unmutated IGHV, do not
respond well to these therapies [10]. CLL treatment has greatly improved with
the development of more specific and targeted agents. Most of these new
agents are currently undergoing clinical trials or have already been approved,
following promising results in the majority of treated CLL cases. This review
first summarizes the available information of these novel agents in CLL, and
then discusses the future incorporation of other new compounds into the
therapeutic armamentarium.

2. NEW TARGETED THERAPIES FOR CLL

2.1. B-Cell Receptor (BCR) Inhibitors

B-cell receptor (BCR) is a transmembrane receptor complex which


incorporates a surface immunoglobulin associated with a signal transduction
moiety (CD79A/B). BCR is indispensable during the normal B cell maturation
process. Signaling through BCR results in distinct outcomes, modulates clone
expansion in a pre-B cell, induces apoptosis of an immature B cell and, in
contrast, encourages the growth and proliferation of mature B cells. Other cell
surface receptors located close to the cell membrane, for example CD 19 and
CD40, are able to modulate BCR signaling in both directions stimuli and
inhibition. A relative restriction of the BCR immunoglobulin heavy chain
variable gene (IGHV) repertoire provides evidence of antigenic selection in
CLL [11, 12]. It has been reported that some CLL samples poorly respond to
BCR stimulation (predominantly IGHV mutated) while others elicit a strong
response to BCR stimulation (predominantly IGHV unmutated). A number of
BCR signaling inhibitors have entered clinical trials in the past few years and
provide a strong rationale for using BCR as a therapeutic target [13, 14].

2.1.1. BTK Inhibition


Brutons tyrosine kinase (BTK) is a non-receptor tyrosine kinase of the
Tec kinase family and plays a crucial role in BCR signaling [15].

2.1.1.1. Ibrutinib
PCI-32765 (Pharmacyclics) is a first-in-class oral covalent inhibitor of
Brutons tyrosine kinase that has been approved for the treatment of CLL
New and Repositioning Approaches to the Treatment 19

patients who have received at least one prior therapy, and as primary therapy
for those with a chromosome 17p13.1 deletion [16, 17]. This agent forms a
bond with the cysteine-481 of BTK [18]. It also inhibits several other kinases,
such as ITK (interleukin-2-inducible T-cell kinase), TEC, BMX, and EGFR.
Ibrutinib has demonstrated promising activity in studies involving relapsed
follicular lymphoma, CLL/SLL, diffuse large B-cell lymphoma and other non-
Hodgkin lymphoma subtypes, either as a single agent or in combination with
other drugs [19-23]. Byrd et al. [24] published results of 101 patients with
relapsed or refractory CLL (RESONATE trial) who received ibrutinib, 34% of
the patients had del(17p), and 78% had unmutated IGHV. The median number
of prior therapies was four, the median age was 64 years. The overall response
rate (ORR) was 90% with a 7% complete remission (CR) and 65% partial
remission (PR). The estimated progression-free survival (PFS) at 30 months
was 69%. For patients with del(17p) and del(11q), the median PFS was 28
months and 38.7 months, respectively, and was remarkably inferior to that of
patients without those aberrations [24]. In a pivotal phase 3 of this trial,
patients with relapsed or refractory CLL were randomized to receive ibrutinib
(n=195) or ofatumumab (n=196). The ibrutinib arm had much higher ORR and
superior PFS and overall survival (OS) as compared to ofatumumab arm [25].
In early-phase data from 31 previously untreated patients with CLL who were
65 years of age or older, ORR with ibrutinib was 84% (with CR in 23% of the
patients); the estimated rate of PFS at 30 months was 96%, and OS rate was
97%, with 81% of the patients continuing to take daily ibrutinib after three
years of follow-up [24]. In phase 3 of RESONATE-2 (NCT01722487), a
multicenter, open-label, randomized trial, the efficacy and safety of single-
agent ibrutinib was compared with those of chlorambucil in patients with
median age of 73 years with previously untreated CLL [26]. During a median
follow-up period of 18.4 months, ibrutinib resulted in significantly longer PFS
than chlorambucil (median not reached vs. 18.9 months), with a risk of
progression or death that was 84% lower with ibrutinib than with
chlorambucil. Ibrutinib significantly prolonged OS rate: at 24 months, the OS
was 98% vs. 85% with chlorambucil, and the OR with ibrutinib was 86% vs.
35% with chlorambucil [26].
It is important to note that most patients develop lymphocytosis after
initiating ibrutinib therefore a novel category of response - PR with
lymphocytosis - was reported. This is assumed to be due to trafficking of CLL
cells from the lymph nodes and other tumor sites into the peripheral blood,
likely due to inhibition of several molecular pathways involved in adhesion,
20 Ida Franiak-Pietryga and Maria Bryszewska

including CXCR4/5 [27, 28]. This problem generally resolves over the course
of between 6-9 months, but about 20% of patients have lyphocytosis over 12
months of ibrutinib treatment [29]. Persistent lymphocytosis is more frequent
in patients with mutated IGHV and in those with del(13q) [27]. Development
of lymphocytosis does not appear to be detrimental to long-term clinical
outcomes [27, 29, 30]. The high activity of ibrutinib in CLL with del(17p) is
remarkable and exceeds the efficacy of high-dose methylprednisone
combinations in this setting [24]. Ibrutinib induced responses in 68% of such
patients, whereas a PFS of 10 months was documented after treatment with
high dose steroids in combination with alemtuzumab [31]. The results of
combining ibrutinib with rituximab [32], ofatumumab [33], ublituximab [34]
or bendamustine [35] in patients with relapsed or refractory CLL high risk
disease including del(17p), and/or del(11q), and/or TP53 mutation look very
promising. However, some preclinical studies have reported antagonism
between ibrutinib and rituximab, probably due to the inhibition of ADCC by
ibrutinib [36-38], the data from these clinical trials (NCT02007044,
NCT02301156, NCT02264574, NCT02048813, NCT01886872) looks very
appealing. Other clinical trials with ibrutinib for treatment-nave and
previously treated CLL patients (RESONATE, HELIOS, RESONATE-2), for
CLL patients with del(17p) (RESONATE-17), for patients with mantle cell
lymphoma (SPARK, SHINE, RAIN), follicular lymphoma (DAWN) continue.
Resistance to ibrutinib has been under study. In several patients who
progress on ibrutinib, a mutation of BTK C481S and gain of function
mutations in PLC2, a signaling molecule causing a downregulation of BTK,
have been found [39]. This mutation reduces the binding affinity of ibrutinib
to BTK, and only allows for reversible BTK inhibition leading to transient
BTK inhibition [40].

2.1.1.2. Spebrutinib
CC-292 (AVL-292; Celgene) is a covalent small molecule Bruton's
tyrosine kinase (BTK) inhibitor and unlike ibrutinib, it does not inhibit the
SRC family kinase or ITK. This drug has indication to treatment not only in
CLL but also in B-cell Non-Hodgkins Lymphoma (B-NHL) and
Waldenstrms macroglobulinemia (WM). A total of 83 patients with relapsed
or refractory CLL were enrolled on a phase 1 study (NCT01351935) i
ntended to evaluate the safety and tolerability of AVL-292 as monotherapy
in subjects with relapsed or refractory CLL, B-NHL or WM [41]. The
most frequent grade adverse events were found to be neutropenia (21%),
New and Repositioning Approaches to the Treatment 21

thrombocytopenia (15%), pneumonia (10%), and anemia (8%). The


recommended phase 2 dose was identified as 500 mg twice a day, and at this
dose, the ORR was 63% (all PR/PR-L) [41]. Eda et al. suggest that based on
these findings, the combination of CC-292 with carfilzomib augments the
inhibitory effects against osteoclasts within the bone microenvironment and
has promising therapeutic potential for the treatment of multiple myeloma and
related bone disease [42]. However, the efficacy and safety of spebrutinib in
CLL remain inclonclusive.

2.1.1.3. Acalbrutinib
ACP-196 (Acerta Pharma) is a novel irreversible second generation BTK
inhibitor which is more selective for BTK than ibrutinib [43]. Acalabrutinib
binds covalently to Cys481 with improved selectivity and in vivo target
coverage compared to ibrutinib and CC-292 in CLL patients [43-45]. In
the in vitro signaling assay on primary human CLL cells, acalabrutinib
inhibited tyrosine phosphorylation of downstream targets of ERK, IKB, and
AKT [46]. Acalabrutinib demonstrated higher selectivity for BTK with IC50
determinations on nine kinases with a cysteine residue in the same position as
BTK [43]. Importantly, unlike ibrutinib, acalabrutinib did not inhibit EGFR,
ITK, or TEC [43, 46]. Compared with ibrutinib, acalabrutinib has much higher
IC50 (>1000 nM) or virtually no inhibition on kinase activities of ITK, EGFR,
ERBB2, ERBB4, JAK3, BLK, FGR, FYN, HCK, LCK, LYN, SRC or
YES1 [46]. The in vivo effects of acalabrutinib against CLL cells have been
demonstrated in a NSG mice model with xenografts of human CLL [47]. The
drug significantly inhibited proliferation of human CLL cells in the spleens of
NSG mice at all dose levels, as measured by the expression of Ki67. The
tumor burden decreased with the treatment in a dose-dependent manner.
Acalabrutinib inhibited BCR signaling by reducing the phosphorylation
of PLC2 and transiently increased CLL cell counts in the peripheral
blood [47]. To further assess the safety, efficacy, pharmacokinetics,
and pharmacodynamics of acalabrutinib in human CLL, a phase 1/2,
multicenter, open-label, and dose-escalation clinical trial has been ongoing
(NCT02029443). In the latest report, 61 patients with relapsed CLL were
enrolled [46]. These patients had relapsed or refractory CLL, 31 % of them
had del(17p) and 75 % had IGHV unmutated. In the phase 1 portion of this
study, patients were treated with acalabrutinib at an increasing dose of 100 to
400 mg once daily. In phase 2, the expansion phase, 100 mg was administered
twice daily. After a median follow-up of 14.3 months, the ORR was 95%, with
22 Ida Franiak-Pietryga and Maria Bryszewska

85% PR and 10% PR-L. The ORR was 100 % in those patients with
chromosome del(17p). The most frequent adverse events included headache,
diarrhea, and weight gain. There were no dose-limiting toxicities observed in
the phase 1 portion of the trial. Acalabrutinib does not inhibit EGFR, thus
could reduce the adverse events on skin rash and severe diarrhea [46].
At this time, a phase 3 study (NCT02477696) commenced comparing
acalabrutinib with ibrutinib in high-risk patients (del(17p), del(11q)) with
relapsed CLL. Additionally, studies in treatment-nave CLL are also being
conducted: (NCT02475681) (obinutuzumab + ACP-196 vs. obinutuzumab +
chlorambucil vs. ACP-196); and ACP-196 in combination with Obinutuzumab
in relapsed or refractory or untreated CLL/SLL/PLL (NCT02296918); and
ACP-196 in patients with relapsed or refractory and treatment-nave deletion
17p CLL/SLL (NCT02337829). A comparison of acalabrutinib with ibrutinib
and other BTK inhibitors will be the only way to confirm the advantages and
disadvantages of these agents.

2.1.1.4. ONO-4059
ONO-4059 (Ono Pharmaceuticals LTD/Gilead Sciences, Inc.) is a highly
potent and selective oral BTK inhibitor, which is more selective for BTK than
ibrutinib. This compound demonstrated anti-tumor activity in pre-clinical
models and in clinical trials in both CLL and NHL patients. ONO-4059
has a favorable safety profile along with promising efficacy over a long
duration in heavily pre-treated CLL, including refractory and currently poor
prognostic del(17p) patients [48]. In an ongoing phase 1 study, ONO-4059
was administered at doses ranging from 20 to 600mg in 25 heavily-pretreated
patients. The treatment was well tolerated, with mostly grade 1-2 adverse
events. Six patients had grade 3-4 neutropenia. The ORR was 84% with a
similar response rate in patients with del(17p); 17 patients had PR and 4 PR-L
for 21 responding patients [48].
In addition to the above mentioned new BTK inhibitors, a few more are
currently undergoing clinical trials: GDC-0834 [49, 50], CGI-560 [51], CGI-
1746 [51], HM-71224 [52], CNX-774 [53], LFM-A13 [54] and RN-486 [55]
(Table 1). All have been developed to target BTK and disrupt the survival of
normal B-cells and their malignant counterparts.
Table 1. Novel BTK inhibitors potentially effective in CLL

Compound Company Indication Clinical trial Mechanism of action


GDC-0834 MedKoo RA, CLL/NHL Early clinical development for the Highly selective, reversible BTK
Biosciences, treatment of B-cell malignancies inhibitor with nanomolar activity in
Inc./Genentech and autoimmune disorders enzyme kinetics studies; IC50 of 5.9
nM. The compound demonstrates
effective activity against BCR- and
CD40-dependent B-cell proliferation
and activation, and potently inhibits
immune complex-mediated
inflammatory cytokine elaboration in
monocytes.
CGI-560 Genentech B-cell and myeloid Early clinical development for the Highly selective (>10 fold) but
cell diseases, RA treatment of B-cell malignancies modestly potent small molecule
and autoimmune disorders inhibitor of BTK with an IC50 of 400
nM in enzymology assays.
CGI-1746 Genentech B-cell and myeloid Early clinical development for the Highly selective small-molecule
cell diseases, RA treatment of B-cell malignancies inhibitor of the Btk with IC50 of 1.9
and autoimmune disorders. nM. is specific for Btk, with 1,000-
CGI1746 abrogates B cell fold selectivity over Tec and Src
dependent arthritis. CGI1746 family kinases.
treatment (100 mg/kg, s.c, twice-
daily dosing) results in significant
inhibition (97%) of overall
clinical arthritis scores.
Table 1. (Continued)

Compound Company Indication Clinical trial Mechanism of action


CGI1746 treatment substantially
reduces TNF, IL-1 and IL-6, as
well as MCP1 and MIP-1 on
both the mRNA and protein level
in the passive anti-collagen II
antibodyinduced arthritis
(CAIA) model. CGI1746 shows
comparable efficacy to TNF
blockade and significantly reduces
clinical scores, as well as joint
inflammation, in mice or rats with
established arthritis.
HM-71224 Hanmi RA, Immune- and The compound has progressed Oral, small molecule BTK inhibitor
Pharmaceuticals autoimmune into phase I clinical testing, and
disease its PD, PK, safety, and tolerability
are being assessed in healthy
volunteers in Korea and the
Netherlands [NCT01765478].
CNX-774 Avila Therapeutics B-cell and myeloid The compound has progressed to Oral, small molecule inhibitor with
cell diseases, RA advanced preclinical development irreversible BTK-inhibitory property.
and additional in-vitro and in-vivo Highly selective for BTK, and forms a
data are awaited. ligand-directed covalent bond with the
Cys-481 residue within the ATP
binding site of the enzyme.
Compound Company Indication Clinical trial Mechanism of action
In biochemical and cellular assays,
CNX-774 demonstrates potent
inhibitory activity towards BTK with
an IC50 of <1nM and 1-10nM
respectively.
LFM-A13 Enzo Life Sciences Leukemia, In human Ph+ ALL-1 and First-in-class, dual BTK/Polo-like
lymphoma, breast NALM-6 pre-B ALL cell line, kinases (PLK) inhibitor with anti-
cancer treatment with LFM-A13 proliferative, pro-apoptotic. A
enhances the sensitivity of the leflunomide metabolite analogue,
cells to both ceramide- and LFM-A13 binds favorably to the
vincristine-induced apoptosis. catalytic site within the kinase domain
Dosed at levels ranging from 10 of BTK, and exhibits an inhibitory
to 80 mg/kg,. potency (IC50) of 17.2microM in cell-
free kinase assays. Highly selective for
BTK, and specifically inhibits cellular
BTK activity human NALM-6
leukemic pre-B cells in a dose-
dependent fashion. >100-fold
selectivity over other protein kinases
including JAK1, JAK2, HCK,
EGFR,and IRK.
RN-486 LKT Laboratories RA, B-cell Preclinical studies also showed Selective BTK inhibitor with IC50 of 4
diseases, SLE that RN-486 efficiently inhibits nM. It blocks BCR-mediated CD69
FcR-mediated TNF- production expression in B-cells in a dose-
in monocytes, and abrogates dependent manner.
FcR-mediated mast cell
degranulation.
Table 1. (Continued)

Compound Company Indication Clinical trial Mechanism of action


Suppresses IgG anti-dsDNA
secretion, blocks CD69
expression in response to BCR
crosslinking, and completely
inhibits progression of glomerular
nephritis in systemic lupus
erythematosus (SLE) prone
NZB/W mouse models.
RA rheumatoid arthritis; CLL chronic lymphocytic leukemia, NHL non-Hodgkin lymphoma; IC50 50% inhibitory concentration; BTK
Brutons tyrosine kinase; BCR B cell receptor.
Source of information: European Medicines Agency (EMA) and US Food and Drug Administration (FDA) reports.
New and Repositioning Approaches to the Treatment 27

2.1.2. PI3 Kinase Inhibition


The PI3K pathway is a key component of survival in many cancers,
including CLL. PI3K, via phosphorylation of the inositol lipid
phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), forms the second
messenger molecule phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)
which recruits and activates proteins containing the pleckstrin homology
domain, leading to downstream signaling events crucial for proliferation,
survival and migration. There are three classes of PI3K isoforms. Class I PI3K
enzymes consist of four distinct catalytic isoforms, PI3K, PI3K, PI3K and
PI3K. The PI3K and PI3K isoforms are expressed predominantly in
leucocytes, whereas the PI3K and PI3K isoforms are ubiquitously expressed
[56]. Of those isoforms, PI3K is abundantly expressed in CLL [57]. PI3K
becomes activated upon ligation of a number of chemokine and cytokine
receptors expressed by CLL cells and following BCR ligation [57, 58]. CLL
patients with more progressive disease (IGHV unmutated) show significantly
greater PI3K expression compared to those with less progressive disease
(IGHV mutated) [59]. Additionally, PI3K activity was found to be higher in
CLL-B cells than in B cells from healthy volunteers. In normal B cells, either
PI3K or PI3K can mediate tonic BCR signaling and low-level AKT
phosphorylation, with PI3K able to compensate for the absence of PI3K and
maintain normal B cell development in the bone marrow. In contrast, agonist-
induced AKT phosphorylation is solely PI3K mediated [56]. Identification of
the hematopoietic-selective isoform PI3K has unlocked a new therapeutic
potential for CLL.

2.1.2.1. Idelalisib
GS-1101 (formerly CAL-101; Gilead Sciences) is an orally bioavailable,
small-molecule inhibitor of PI3K. Idelalisib has recently gained approval for
the treatment of relapsed/refractory CLL. It has been evaluated in a phase I
clinical trial in 54 CLL patients with relapsed/refractory disease; nodal
shrinkage and overall survival were obtained in 81% and 72% patients
respectively [60]. Idelalisib has shown considerable monotherapy activity
when given at dose levels of 100mg or higher twice daily in patients with
heavily pretreated indolent non-Hodgkins lymphoma. About one-fourth of the
patients had del(17p), and 91% had unmutated IGHV. The median number of
prior therapies was five. In the dose escalation phase, six dose levels of oral
idelalisib (50-350 mg once or twice daily) were tested. The ORR was 72%
(39% PR, 33% PR-L). The median PFS was 15.8 months. The most commonly
noted grade 3 adverse events were pneumonia (20%), neutropenic fever
28 Ida Franiak-Pietryga and Maria Bryszewska

(11%), and diarrhea (6%) [60]. Idelalisib was found to demonstrate substantial
activity in older, treatment-nave patients with CLL when used as a single
agent [61].
In a phase III clinical trial, idelalisib combined with the anti-CD20
antibody rituximab significantly improved progression-free survival (81%)
and overall survival (91%) in relapsed CLL patients (n = 220) compared to
placebo plus rituximab [62]. In this study, combination therapy achieved a
response and survival benefit compared with rituximab alone [62]. The
patients receiving idelalisib and rituximab demonstrated improved rates of OR
(81%) in comparison with those with rituximab monotherapy (13%). Median
PFS was 5.5 months in the rituximab arm, but was not reached in the idelalisib
combined with rituximab group. Moreover, OS at 12 months was 92% for
idelalisib combined with rituximab, compared to 80% for rituximab alone
[62]. Idelalisib combined with rituximab therapy is also very effective in
patients with high risk CLL (with del(17p), TP53 mutations, del(11q)) [63].
Another phase 3, randomized study evaluating the efficacy and safety of
idelalisib with ofatumumab for previously-treated CLL patients is in progress
(NCT01659021).
Idelalisib demonstrates a dual mechanism of action by inhibiting pro-
survival signaling pathways [57] and, like other kinase inhibitors, by blocking
ingress into and promoting egress out of the lymph node into the blood,
leading to the re-localisation of tumour cells. Release from the protective
lymph environment into the blood renders CLL cells more susceptible to
apoptosis. PI3K is expressed by all leucocytes including T cells, raising the
possibility that the therapeutic effect of idelalisib may, at least in part, be due
to effects on the surrounding immune cells in addition to direct effects on CLL
cells [64]. Intriguingly, IL-4 protects against idelalisib-induced apoptosis in
vitro [57], indicating that microenvironmental influences may protect CLL
cells against PI3K inhibitors and that co-inhibition of the function of
surrounding cells may be an important factor in successful treatment. Ongoing
clinical trials with idelalisib are examining the combination with other agents;
including rituximab, ofatumumab, obinutuzumab and bendamustine.
Furthermore, a recent publication has shown that a combination of idelalisib
with ibrutinib is synergistic, indicating potential benefit from combined or
sequential therapy [65]. In addition to idelalisib, the development of other
PI3K inhibitors for the treatment of lymphoid malignancies is ongoing: one
such example being TGR-1202, a novel PI3K inhibitor with significant
differences in its chemical structure compared to idelalisib and with lower
reported incidences of colitis in patients. TGR-1202 is currently in phase I
New and Repositioning Approach to Treatment 29

clinical trials, with significant nodal responses observed in 88% of


relapsed/refractory CLL patients to date (NCT01767766).

2.1.2.2. Duvelisib
IPI-145 (Infinity Pharmaceuticals, Inc) targets both PI3K and PI3K
isoforms [66] and induces apoptosis in CLL samples in vitro, abrogates bone
marrow stromal cell-mediated survival, and inhibits BCR-mediated signaling
and chemotaxis in response to CXCL12 [67]. It is very important that
duvelisib also killed CLL cells that were resistant to ibrutinib [68], so this may
hold true with other PI3K inhibitors, and could form an important strategy for
treating patients refractory to ibrutinib. In a phase 1 study, 55 patients with
relapsed or refractory CLL received duvelisib orally at doses from 8 mg to 75
mg twice daily. ORR was 58% with a CR rate of 2%. The PFS at 18 months
was 60%. In patients with del(17p), the median PFS was 14 months, while
89% of patients showed a reduction (50%) of enlarged lymph nodes [67, 68].
The most commonly noted grade 3-4 adverse event was neutropenia (42%).
Duvelisib is now in a number of clinical trials for CLL, including those
investigating its use in combination with anti-CD20 antibodies or
bendamustine, and in patients refractory to ibrutinib (NCT02292225,
NCT01871675, NCT02576275).

2.1.2.3. TGR-1202
TGR-1202 (formerly known as RP5264; TG Therapeutics) is an orally
available PI3K inhibitor with once-daily dosing, targeting the delta isoform
with nanomolar potency and several-fold selectivity over the alpha, beta, and
gamma isoforms of PI3K. Inhibition of PI3K delta signaling with TGR-1202
has demonstrated robust activity in numerous pre-clinical models and primary
cells from patients with hematologic malignancies. OConnor et al. [69] report
updated safety and efficacy results from a phase I study of TGR-1202 in
patients with relapsed and refractory CLL and lymphoma. TGR-1202 is
administered orally once-daily (QD) following a 3+3 dose escalation design.
Expansion cohorts were open at 800 mg, 1000 mg, and 1200 mg QD. Of 16
evaluable CLL patients, 15 (94%) achieved a nodal PR (median nodal of
76%), of which 10 (63%) achieved a PR according to the Hallek et al. [70]
criteria. Among the 32 evaluable NHL patients, 10 achieved an objective
response, including 3/11 evaluable patients with DLBCL, while responses
have been limited in patients with MCL (1/5) and HL (1/9). Of the 16
evaluable indolent NHL (FL & MZL) patients, 14 (88%) have achieved
reductions in tumor burden with six patients on study for over 12 cycles (and
30 Ida Franiak-Pietryga and Maria Bryszewska

durations upwards of 29+ cycles), with 5/12 FL and 1/4 MZL patients
achieving an objective response to date [69]. Burris et al. [71] reported the
data of 66 patients with NHL (20 CLL) enrolled to the study. The ORR in
patients with CLL was 63%. The most commonly noted grade 3-4 adverse
events were neutropenia (11%), anemia (8%), skin rush (5%), dyspnea (5%),
and fatigue (3%). TGR-1202 has been combined with ublituximab in patients
with relapse or refractory NHL, including CLL [72]. In total, 55 patients were
included to the trial. Infusion-related reaction was seen in 29% patients. ORR
in the CLL cohort was in 83% patients (all PR) was noted. In a preliminary
analysis of 16 patients with relapse and refractory NHL, the triplet
combination of TGR-1202, ublituximab and ibrutinib has been evaluated
(NCT02006485) and the triplet was safe with about 90% nodal response [73].

2.1.3. Pan-PI3K/mTOR Inhibition


mTOR is crucial for cellular proliferation and is an attractive therapeutic
target; however, the use of selective mTOR inhibitors is hampered by
disruption of negative feedback mechanisms via S6 kinase and mTORC2 and
subsequent over-activation of PI3K and AKT, respectively. Dual PI3K/mTOR
inhibitors may overcome this effect by directly inhibiting mTOR and
preventing over-activation of AKT via simultaneous inhibition of PI3K. As
mTOR can also be activated by MAPK mediated signaling, the inhibition of
PI3K in combination with inhibition of mTOR may lead to greater suppression
of this pro-survival pathway than PI3K inhibition alone [74]. This is
demonstrated by a disconnect between PI3K/AKT activity and
phosphorylation of the mTOR target 4E-BP1 in CLL [75], which indicates that
inhibition of PI3K alone would not achieve maximal inhibition of mTOR
mediated signaling. Blunt et al. [76] note that CLL cells treated with the dual
PI3K/mTOR inhibitor PF-04691502 or idelalisib plus mTOR inhibitor
everolimus, triggered apoptosis to a significantly greater degree compared to
PI3K or mTOR inhibition alone in an in vitro study. Another substance, PF-
04691502, also inhibited chemotaxis, reduced BCR stimulated
signaling/survival and significantly prolonged survival in a murine model of
CLL [76]. Various PI3K/mTOR inhibitors are progressing through clinical
trials for a number of different cancers and may therefore provide a promising
novel treatment strategy for CLL and other B cell malignancies.
Pictilisib (GDC-094; Piramed LTD/Genentech, Innc.) is a highly-specific
PI3K inhibitor of class I PI3Ks with some activity against mTOR [77]. The
drug was examined in solid tumors and NHL (NCT00876122) It also has anti-
proliferative and pro-apoptotic properties, and is active against primary T cell
New and Repositioning Approach to Treatment 31

acute lymphoblastic leukemia (T-ALL) [78]. Although Pictilisib has not been
investigated in CLL yet, it has a great potential for antileukemic activity.

2.1.4. SYK Inhibition


Spleen tyrosine kinase (SYK), an intracellular tyrosine kinase, plays a
central role following the activation of cells by B or T cell receptors (BCR or
TCR), cytokines or adhesion molecules [79-81]. SYK activation is mediated
when sarcoma kinases (Src kinases) phosphorylate conserved sequences
within receptors containing immune tyrosine activation motifs (ITAMs) [82,
83]. Once activated, SYK propagates the phosphorylation of further
downstream molecules including the PI3-K and MAP-kinase pathways [84,
85]. SYK and the zeta-chain-associated protein kinase 70 (ZAP-70) are the
only kinases of the SYK family. SYK initiates downstream signaling and
amplifies the original BCR signal. Preclinical studies established SYK as a
valid therapeutic target in CLL [86-88].

2.1.4.1. Fostamatinib
R788, an oral pro-drug of the active metabolite R406 (Rigel
Pharmaceuticals) is an ATP-competitive kinase inhibitor that also inhibits a
number of other kinases [89]. In vitro treatment of CLL cells with fostamatinib
inhibits BCR and integrin signaling, antagonizes the protective effect of
stromal cells, reduces migration to chemokines and adhesion to stromal
components, and induces a moderate degree of apoptosis [86]. Fostamatinib
prevents disease progression both in TCL1 transgenic mice, in which antigen-
dependent selection appears to play a similar role as in human CLL, and in a
non-Hodgkin lymphoma model that depends on cooperation between MYC
and BCR-derived signals [88, 90]. A group of 68 patients with relapsed and
refractory NHL and CLL were enrolled to a phase 1 study with fostamatinib as
monotherapy [91]. In this group, 11 patients had CLL and 55% achieved PR.
ORR of 10% was achieved in the follicular lymphoma cohort, and 11% in the
mantle cell lymphoma cohort. The dose-limiting toxicity was diarrhea,
neutropenia and thrombocytopenia. Further clinical phases in CLL had been
stopped due to negative phase 3 results in patients with rheumatoid arthritis.
This drug is currently being tested in immune thrombocytopenic purpura (ITP)
[92].

2.1.4.2. Entospletinib
GS-9973 (Gilead Sciences) is an oral inhibitor of SYK which is more
selective than R406 [93]. Entospletinib is currently being pursued in phase 2
32 Ida Franiak-Pietryga and Maria Bryszewska

clinical trial with patients with relapsed or refractory CLL (41 patients) and
NHL (145 patients). Patients received the drug 800 mg orally twice daily. In
the CLL cohort, ORR was achieved in 61% (all PR), with a median PFS of 14
months. Among adverse events, dyspnea, pneumonia, febrile neutropenia,
dehydration, and pyrexia were the most commonly noted [94].

2.1.5. LYN Inhibition


The SRC family nonreceptor tyrosine kinase LYN initiates BCR signaling
by phosphorylating activation motifs on the Ig and Ig chains that recruit
further components of the signaling pathway. LYN not only phosphorylates,
and thereby activates, SYK, but it also activates phosphatases that in turn
inhibit signal transduction through the BCR [95]. LYN thus controls both
activation as well as termination of BCR signaling. Mice deficient in LYN
have reduced numbers of B cells [96]. An essential function of LYN is to
down-regulate BCR activation and limit the expansion of autoreactive B cells.
It is also reported that CD79B mutations in ABC-DLBCL reduce LYN kinase
activity and promote chronic active BCR signaling, resulting in constitutive
NF-B activation [97].

2.1.5.1. Dasatynib
Dasatinib (Bristol-Myers Squibb) is an oral multikinase inhibitor targeting
SRC and ABL kinases which is approved for use in imatinib-resistant chronic
myeloid leukemia (CML). Recently, it has been shown that dasatinib not only
inhibits LYN but also BTK at low nanomolar concentrations [98]. Reports
from in vitro studies revealed that dasatinib induces apoptosis in CLL cells
with no correlation between response and inhibition of LYN phosphorylation.
However, induction of apoptosis in vitro was inversely correlated with drug-
induced inhibition of SYK phosphorylation. Dasatinib inhibited BCR
signaling, stromal cell contact and CD40 stimulation causing an antagonized
proapoptotic effect [99]. A group of 15 patients with relapsed or refractory
CLL were enrolled in a phase 2 study where they were administered of 140 mg
dasatinib once daily. The OR rate was 20% with a PFS of 7.5 months, and five
patients showed a reduction (50%) of enlarged lymph nodes.
Myelosuppression was the primary toxicity, with grade 4 neutropenia and
thrombocytopenia occurring in 40% and 13% of patients, respectively [100].
New and Repositioning Approach to Treatment 33

2.2. BCL2 Inhibitors

The BCL-2 family members are key regulators of the intrinsic apoptotic
pathway and are classified into three classes of proteins based on their
structural similarity and function: the anti-apoptotic proteins (BCL-2, BCL-xL,
MCL-1, BFL-1, and BCL-w) sequester the BH3-only proteins (BIM, BID,
PUMA, and NOXA) that in turn activate the pro-apoptotic proteins (BAX and
BAK). In some cases, anti-apoptotic BCL-2 proteins can also sequester pro-
apoptotic proteins. BAX/BAK oligomerization causes mitochondrial outer
membrane permeabilization, resulting in cytochrome c release and apoptosis
[101]. The dysregulation of BCL2 family proteins results in the inhibition of
apoptosis and uncontrolled proliferation of B-cells, leading to the development
of many hematological malignancies and contributing to the development of
resistance to chemotherapy. Therapeutic modulation of the BCL2 pathway
suggests the possibility of a promising new therapeutic strategy in diseases
with impaired mechanism of apoptosis, for example CLL and NHL [102].

2.2.1. Venetoclax
ABT-199 (GDC-0199, RG7601; Genentech/AbbVie) is a small-molecule
designed with greater selectivity for BCL2 [103]. The agent binds more avidly
to BCL2 than to BCL-XL by >3 orders of magnitude [104]. It binds to BCL-
XL and BCL-W but not to MCL1. The affinity of this agent for BCL2 is more
than 500 times higher than that of BCL-XL. Venetoclax is a very promising
agent in the future therapy of CLL. It induces apoptosis in CLL cells at
concentrations 10-fold lower than those required by navitoclax. However, a
200-fold higher concentration of venetoclax was required for induction of
platelet apoptosis compared with navitoclax. Unfortunately, venetoclax also
induces the tumor lysis syndrome (TLS), in which the debris of dying tumor
cells circulating in blood and affect other organs [105]. Preliminary reports
note an ORR of 77% and a CR rate of 23% in groups of patients with relapsed
or refractory CLL following venetoclax monotherapy [106]. The estimated
median PFS was 18 months. It is significant that the CR rate was higher than
that observed following the use of BCR inhibitors in relapsed or refractory
CLL. Venetoclax in combination with rituximab in 49 patients was associated
with a CR rate of 41%, with 50% of patients demonstrating MRD-negative
remission [107]. Two trials are ongoing: one is a randomized phase 3 study for
previously untreated CLL patients venetoclax + obinutuzumab vs.
chlorambucil + obinutuzumab (NCT02242942), and the other for relapsed or
34 Ida Franiak-Pietryga and Maria Bryszewska

refractory CLL patients comparing venetoclax + rituximab vs. BR


(NCT02005471). Cervantez-Gomez et al. [108] report synergy between
venetoclax and ibrutinib.

2.2.2. Navitoclax
ABT-263 (Abbott Laboratories) is a first generation, NOXA-like BH3
mimetic developed for apoptosis-based therapy for CLL. It inhibits BCL2
family members and binds with high affinity to BCL-XL, BCL2 and BCL-W
but not MCL1 or A1 [109, 110]. During a phase 1 study including 26 CLL
patients with relapsed or refractory disease, 35% patients demonstrated PR and
an additional seven patients had stable disease for > 6 months. Median PFS
was calculated for 25 months. The benefits of navitoclax have been observed
in patients with fludarabine refractory CLL, bulky lymphandenopathy and
CLL with del(17p) [111]. The most frequent adverse event was reported as
thrombocytopenia due to BCL-XL inhibition.

2.2.3. Obatoclax
GX15-070 (Cephalon) is an inhibitor of the BCL2 family of proteins. This
inhibition induces apoptosis in cancer cells, preventing tumor growth. It is in
phase 2 clinical trials for the treatment of leukemia, lymphoma, myelofibrosis
and mastocytosis. OBrien et al. [112] reported a phase 1 study with obatoclax
mesylate (NCT00600964). The drug was administered to patients with
advanced CLL at doses ranging from 3.5 to 14 mg/m2 as a one-hour infusion
and from 20 to 40 mg/m2 as a three-hour infusion every three weeks. Twenty-
six patients received a total of 74 cycles. Dose-limiting reactions were
neurological (somnolence, euphoria, ataxia) and associated with the infusion.
The maximum tolerated dose (MTD) was 28 mg/m2 over three hours every
three weeks. One (4%) of the 26 patients achieved a partial response. Patients
with anemia (3/11) or thrombocytopenia (4/14) experienced improvements in
hemoglobin and platelet counts. Circulating lymphocyte counts were reduced
in 18 of 26 patients with a median reduction of 24%. Activation of Bax and
Bak was demonstrated in peripheral blood mononuclear cells, and induction of
apoptosis was related to overall obatoclax exposure, as monitored by the
plasma concentration of oligonucleosomal DNA/histone complexes.
Obatoclax mesylate has biological activity and modest single-agent activity in
heavily-pretreated patients with advanced CLL.
New and Repositioning Approach to Treatment 35

2.3. NF-kB Targeting Agents

A critical role is played in CLL Tumor by B-cell activating factor receptor


(BAFF-R) and CD40, two members of the necrosis factor receptor (TNFR)
superfamily. CD40L-mediated activation of members of the anti-apoptotic
BCL2 family (BCL2, BCL-XL, MCL1 and BFL1) and downregulation of pro-
apoptotic BH-3-only proteins is a well-known phenomenon [113, 114].
Recruitment of the TNFR-associated factor (TRAF) proteins by TNFR family
members leads to activation of the NF-B pathways. NF-B mediates
crosstalk between BCR and TNFR signaling [115]. Activation of the proximal
BCR signaling complex ultimately results in activation of IKK1/IKK2,
phosphorylation and proteosomal degradation of IB, and induction of the
canonical NF-B signaling, resulting in the upregulation of a number of
chemokine molecules (CCl17, CXCR5), cell cycle regulators such as Cyclin D
and the anti-apoptotic proteins mentioned above. Hence, the NF-B pathway
represents an attractive target in CLL.
Inhibition of NF-B in vitro has been proved to result in apoptosis of CLL
cells [116, 117]. NF-B activity might be targeted by proteasome inhibitors
such as bortezomib (Janssen-Cilag) and carfilzomib (Onyx Pharmaceutical)
due to disrupted degradation of IB. Bortezomib has been successfully
applied in vivo to boost the activity of the R-CHOP regimen in activated B-cell
diffuse large B-cell lymphoma, a subtype characterized by the constitutive
activation of NF-B [118]. The FDA has approved this drug for second line
treatment of mantle cell lymphoma, where the disease follows a similar
mechanism of action [119]. The results of an in vitro [120] study of
bortezomib on CLL cells did not confirm the in vivo results of a clinical trial
on a group of fludarabine-refractory CLL patients [121]. The inhibition of
bortezomib in vivo in CLL has been attributed to dietary flavonoids, quercetin
and myricetin identified in the plasma of patients. Such lack of activity has not
been observed in myeloma cells [122]. Yun et al. [123] examined the efficacy
and safety of rituximab and bortezomib in relapsed or refractory indolent B
cell non-Hodgkin's lymphoma (NHL) using the following treatments:
rituximab 375 mg/m(2), i.v. on days 1, 8, 15, and 22 of cycle 1 and on day one
of cycles 2-5, bortezomib 1.6 mg/m(2), given by intravenous injection on days
1, 8, 15, and 22 of a maximum of five cycles. Sixty successive patients were
enrolled in the study over two years at Tianjin cancer hospital lymphoma
department. All patients were recurrent or refractory indolent B cell NHL,
including follicular lymphoma grades 1-2 (n = 35), small lymphocytic
lymphoma/chronic lymphocytic leukemia (LL/CLL; n = 16) and marginal
36 Ida Franiak-Pietryga and Maria Bryszewska

zone lymphoma (n = 9). The median follow-up time was 30 months (range 12-
48). The overall response rate was 70.0 %, with a CR rate of 31.7 %. The 2-
year OS and PFS of all patients were 75.0 and 41.0 %, respectively. RB
chemotherapy in patients with refractory or relapsed indolent B cell NHL was
effective with low toxicity [123].
Carfilzomib irreversibly blocks the release of NF-B to the nuclei thus
abrogating pathway activity [124]. This drug has demonstrated promising pre-
clinical activity in CLL and had little effect on baseline NF-B activity in
peripheral CLL cells [125]. However, NF-B is significantly more active in
the lymph node microenvironment [126]. The findings of a phase 1 study
indicate that carfilzomib provided stable disease in four patients with FL and
one patient with CLL/SLL [127].
To determine the safety and tolerability of carfilzomib in
relapsed/refractory CLL or small lymphocytic lymphoma (SLL), a phase 1
dose-escalation trial including nineteen patients treated with carfilzomib was
performed. The initial dose of 20 mg/m2 was escalated in four cohorts (27, 36,
45 and 56 mg/m2) on days 1, 2, 8, 9, 15 and 16 of 28-day cycles. Therapy was
generally well tolerated, and no dose limiting toxicities were observed. The
most common hematologic toxicities were thrombocytopenia and neutropenia.
All patients evaluable for response had stable disease, including patients with
del(17p) and fludarabine-resistant disease [128].
Other proteasome inhibitors such as MLN9708 (Millenium
Pharmaceuticals), CEP-18770 (Teva/Cephalon) and orpozomib (Onyx
Pharmaceuticals) are currently being investigated in phase 1 protocols in
patients with relapsed or refractory B-cell lymphomas.
The huge group of monoclonal antibodies (mAbs) offers promise. CD20 is
a well-established target for mAbs in CLL, and several other targets
including CD52, CD23, CD19 and CD37 have also been investigated. In
addition, further innovative new therapies are possible with incorporate
immunomodulatory drugs (lenalidomide), cyclin dependent kinase (CDK)
inhibitors (flavopiridol, dinaciclib, TG02), chimeric antigen receptor (CAR) T
cell therapy and novel strategies based on exportin-1 and selinexor. The use of
mAbs and other novel drugs in the treatment of CLL is so expansive that
deserves a separate publication.
Targeted therapies for CLL as outlined above have significantly changed
the treatment paradigm for patients with CLL. Several of these agents have
gained FDA approval and are commercially available. The greatest challenge
at this time is how best to incorporate these novel therapies in the clinical care
of patients.
New and Repositioning Approach to Treatment 37

3. REPOSITIONING DRUGS TO CLL TREATMENT


The introduction of a new drug on the market costs about a billion dollars.
In addition, the time between starting research and the appearance of the drug
on the market is typically 15-17 years. These estimates are of course based on
the traditional methods of searching for new drugs based on phenotypic
screening or searching for a substance, which affects the specific purpose. By
using phenotypic screening, the need for testing the activity is avoided and
instead the research process focusses on the search for a substance with a
specific phenotypic effect. In any case, after identifying the so-called leading
compound, which is the starting structure exhibiting activity, further study
and optimization is needed to increase its activity and to avoid serious side
effects.
Unfortunately, despite our extensive knowledge of the mechanisms of the
course of many diseases and the huge amount of drugs used for various
diseases, finding effective treatment is still a challenge. Therefore, newer
methods of screening are being developed to identify additional uses of
existing active substances. One such method is the repositioning of drugs, in
which the key role is played by databases and statistical analyses. New
discoveries are now possible thanks to the fact that a single molecule can act
on multiple targets. It is estimated that by using efficient repositioning
methods, it is possible to shorten the entry time of new therapies to 3-12 years,
as the safety of existing drugs is already known, allowing them to enter
clinical trials much more quickly.

3.1. Metformin

1,1-Dimethylbiguanide hydrochloride (metformin), the most widely


prescribed oral hypoglycemic agent, which is inexpensive and well tolerated,
has recently received increased attention for its potential antitumor activity.
Recent studies have provided evidence that diabetic patients receiving
metformin have a reduced risk of developing cancer and decreased cancer
mortality [129, 130]. Metformin reduces tumor growth not only indirectly
(systemic effect: glucose and insulin lowering) but also by direct inhibition of
the energy metabolism [131] and inhibition of pathways involved in cell
proliferation [131-133], through both AMPK-dependent [134, 135] and
AMPK-independent mechanisms [136, 137]. Metformin inhibited the
activation of transcription factors based on CLL pro-survival and pro-
38 Ida Franiak-Pietryga and Maria Bryszewska

activation pathways. Particularly involved in CLL disease progression are


PI3/Akt [138] and NF-B/STAT3. Induction of cell death is associated with
inhibition of MCL1, a BCL2 family survival protein which is highly relevant
in CLL. Metformin-mediated down-regulation of this protein did not appear to
be only caused by global translational inhibition, as observed for MCL1 in
metformin-treated head and neck squamous cell carcinoma cell lines [139],
since MCL1 reduction was associated with increased NOXA expression.
Cytotoxic effects of metformin were previously reported in two studies
performed on unstimulated quiescent CLL cells [140, 141]. Both studies
attributed this effect to inhibition of oxidative phosphorylation. Bruno et al.
[142] provide evidence that metformin also modulates glycolytic capability on
CLL cells stimulated to enter the cell cycle. Metformin was administered in
vitro either to quiescent cells or during CLL cell activation stimuli, provided
by classical co-culturing with CD40L-expressing fibroblasts. At doses that
were ineffective on normal lymphocytes, metformin induced apoptosis of
quiescent CLL cells and inhibition of CLL cell cycle entry when stimulated by
CD40-CD40L ligation. This cytostatic effect was accompanied by decreased
expression of survival- and proliferation-associated proteins (MCL1, BCL2),
inhibition of signaling pathways involved in CLL disease progression and
decreased intracellular glucose available for glycolysis. Metformin also
induced the expression of NOXA, a pro-apoptotic protein. In addition, the
team analyzed the effects of metformin on the intracellular expression of
Akt(pan), STAT3(pan) and the phosphorylated forms pSTAT3(Tyr705),
pSTAT3(Ser727), pAkt(Ser473), and pNF-kB(Ser536). Voltan et al. [143]
observed that metformin alone exhibited dose-dependent anti-leukemic
activity in both B leukemic cell lines and primary B-chronic lymphocytic
leukemia (B-CLL) patient cells, and this anti-leukemic activity was enhanced
when used in combination with DCA (dichloroacetate). This innovative
treatment (Met-DCA) induced synergistic apoptotic cell death combined with
a marked down-modulation of MCL1 protein. Although recent observations
are promising, clinical trials are required to determine whether this medication
has the potential to become an adjuvant to current CLL therapies. One pilot
study of metformin mono-therapy in poor prognosis CLL patients began in
2012 (NCT 01750567).
New and Repositioning Approach to Treatment 39

3.2. Statins

Cholesterol plays an important role in cancer development [144-146],


drug resistance and chemoimmuno-sensitivity. Statins lower cholesterol levels
by inhibiting the action of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-
CoA) reductase, an enzyme which catalyzes the conversion of HMG-CoA into
mevalonate, a rate-limiting step in the cholesterol biosynthetic pathway [147].
They can induce apoptosis through extrinsic and intrinsic pathways [148, 149],
but also negatively interfere with CD-20 and rituximab-mediated activity.
Benakanakere et al. [150] has reported that MEC-2 cells (CLL) treated
with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475)
reduced 20% of total cellular cholesterol levels, but also significantly
promoted CD-20 surface expression. Furthermore, treatment of cells with
fludarabine, rituximab or their combination in the presence of BIBB-515,
YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity
measured by cell viability. Moreover, these cholesterol-lowering agents also
significantly enhanced the chemoimmuno-sensitivity of PBMCs taken from
CLL patients.
Chapman-Shimshoni et al. examined the mechanisms of action of
simvastatin and its effects on malignant B cells derived from patients with
CLL. Purified B-CLL cells from 15 patients were cultured either alone or with
simvastatin at concentrations of 10, 50, and 100 M. The level of apoptosis,
measured by annexin binding to exposed phosphatidylserine moieties,
increased significantly in the treated cells at concentrations higher than 50 M
for 24 hours. The apoptosis induced by simvastatin was probably initiated by
mitochondrial caspase 9, which indirectly leads to activation of caspase 3 and
8 [151].
Podhorecka et al. [152] revealed that simvastatin simultaneously induced
apoptosis of CLL cells and reduced their BCL-2/BAX ratio. They also note
that its pro-apoptotic effect is tumor-specific; it does not affect normal
lymphocytes. It was also observed that combining simvastatin with fludarabine
or cladribine resulted in a synergic effect in inducing apoptosis. The results
suggest that simvastatin can be used in the treatment of CLL patients as a
single agent as well as in combination with purine analogs, being equally
effective both in high-risk and good-prognosis patients. Rigoni et al. [153]
found that the mevalonate pathway-dependent Ras/ERK1-2 and RhoA/RhoA
kinase signaling cascades, and the downstream HIF-1/P-glycoprotein
axis were more active in unmutated IGHV than mutated cells,
providing constitutive protection from doxorubicin-induced cytotoxicity. The
40 Ida Franiak-Pietryga and Maria Bryszewska

constitutive MDR phenotype of IGHV unmutated cells was partially


dependent on B cell receptor signaling, as shown by the inhibitory effect
exerted by ibrutinib.
Atorvastatnin has also drawn a great deal of research attention as a
promising anti-leukemic drug. An in vitro study based on CLL cells retrieved
from patients conducted by Zolnierczyk et al. [154] found the cells to be
extremely sensitive to atorvastatin. Apoptosis was found to be induced in
the drug-treated model cells, and this was confirmed by the reduction or
proteolysis of apoptotic markers such as PARP-1, lamin B and p27Kip1,
increased numbers of sub-G1 cells and greater DNA ladder formation. During
atorvastatin-triggered apoptosis, changes were also observed in the expression
of proteins of the BCL2 family. Atorvastatin was also found to have promising
pro-apoptotic results by Yavasoglu et al. [155], who also report accompanying
decreases in the expression of CD5 and ZAP-70.

3.3. Danazol

Danazol is an attenuated synthetic hormone with weak androgenic effects.


It has unique properties similar to corticosteroids and a structure related to
testosterone and ethisterone. The drug is widely used in the treatment
of endometriosis and mammary gland dysplasia. In hematology, danazol
is used with success in immune thrombocytopenic purpura, autoimmune
hemolytic anemia [156, 157] but also in myeloproliferative neoplasms and
myelodysplastic syndromes [158, 159].
Podhorecka et al. report the induction of apoptosis under the influence of
danazol, both used as monotherapy and in combination with the purine
nucleoside analogs fludarabine, cladribine or bendamustine, in leukemic cells
obtained from the peripheral blood and bone marrow of 23 CLL patients
[160]. After 24 hours of incubation, significant apoptosis was observed, as
indicated by active caspase-3 expression and cytotoxicity. Danazol had a
caspase-dependent (Caspase 3, 8, 9) pro-apoptotic and cytotoxic effect on
leukemic cells in a tumor-specific manner. It demonstrated a synergic effect
with cladribine, an additive effect with fludarabine, and an infra-additive effect
with bendamustine. The rate of danazol-induced apoptosis and cytotoxicity did
not differ between patients with better and worse prognostic markers [160].
New and Repositioning Approach to Treatment 41

3.4. Valproic Acid

Valproic acid (VPA) is a short-chain fatty acid that belongs to the group
of histone deacetylase inhibitors (HDAC-I), a relatively new class of agents
used for anticancer therapy. VPA has been used as an anticonvulsant and
mood-stabilizing drug for decades. Even when taken over a long time, VPA is
usually well-tolerated, although it is contraindicated during pregnancy because
of its teratogenic effects [161]. HDAC-Is have been found to exert pleiotropic
antitumor effects by inducing growth arrest, differentiation, and apoptosis,
under both in vitro and in vivo conditions [162]. In malignant cells, HDACIs
induce apoptosis by the upregulation of pro-apoptotic genes and repression of
antiapoptotic genes [163, 164]. Bokelmann and Mahlknecht [165] observed
that VPA, mediates apoptosis in CLL cells ex vivo by caspase activation via
both the extrinsic and the intrinsic apoptosis pathways, as indicated by the
activation of the caspase proteins 8 and 9, and cleavage of the proapoptotic
protein BID. VPA treatment reduced BCL2 mRNA levels, resulting in a lower
BCL2/BAX ratio. A year later, Bouzar et al. [166] reported that VPA acted in
a highly synergistic/additive manner with fludarabine (FA) and cladribine
(2CdA) to induce apoptosis of B-CLL cells. Moreover, VPA also restored
sensitivity to fludarabine in B cells from high-risk CLL patients.
The mechanism of apoptosis induced by VPA, either alone or combined
with FA or to 2CdA was caspase-dependent and involved the extrinsic
pathway. VPA stimulates hyperphosphorylation of p42/p44 ERK, cytochrome
c release and overexpression of BAX and FAS. Yoon et al. [167] confirm the
presence of a synergistic cathepsin B mediated activation of the apoptotic
response between VPA and FA in a CLL patient in vivo, leading to a decrease
in the anti-apoptotic proteins. They also found that the combination of FA and
VPA decreases the level of the anti-apoptotic proteins MCL1 and XIAP in
primary CLL cells. Moreover, the combined therapy resulted in reduced
lymphocyte count in five out of six patients, and reduced lymph node sizes in
four out of six. Karp et al. [168] report a VPA median cytotoxicity of 13.88%
(range 0-54.65%) in a study based on peripheral blood mononuclear cells
retrieved from 53 patients with CLL. It has been proved that the effect of VPA
on CLL cells is dependent on lactate dehydrogenase serum levels, but is
independent of all other prognostic markers. Considering resistance to therapy,
NOTCH1 mutations have been checked. It is belived that CD20 levels are low
in CLL, which may be responsible for the dysregulation of HDAC-mediated
epigenetic repression of CD20 expression [169]. This may prove to be
valuable information for future targeted therapy in CLL.
42 Ida Franiak-Pietryga and Maria Bryszewska

4. NANOPARTICLE BASED THERAPEUTICS APPROACH TO


CLL TREATMENT
Highly flexible, fast drug design and production based on tumor genetic
profiles, which also enables much more intensive and effective drug selection
for individual treatment of patients, has been made possible by the discovery
of novel nanomedicines. Nanomedicine, as a discipline, offers key advantages
in overcoming cancer drug resistance. The advanced design and alternative
mechanisms of drug delivery possessed by such nanodrugs as liposomes,
polymer conjugates, micelles, dendrimers, carbon-based nanodrugs, and
metallic nanoparticles offer great promise for overcoming numerous forms of
multi-drug resistance and may open new horizons for cancer treatment.

4.1. Dendrimers

Dendrimers are highly branched, perfectly monodisperse macromolecules


with a precisely controlled chemical structure. The globular shape of
dendrimers is a result of their internal structure, in which all bonds emerge
radially from a central core with repeat units. This generates branching points,
which allows at least two monomers to be attached [170]. Due to their
nanometric size, dendrimers can interact effectively and specifically with cell
components such as membranes, organelles, proteins and nucleic acids [171-
173]. Our research team has been working on the use of poly(propylene imine)
dendrimers (PPI) as potential agents for CLL. Dendrimer surface modification
seems to be one of the best ways to reduce their toxicity; one such
modification being sugar residues. Our former studies indicate that PPI
dendrimers partly modified with maltotriose (PPI-G4-OS, where OS stands for
open shell,) are toxic to selected cancer cells (U87, CEM-SS, MEC1)
whereas their fully-modified counterparts (PPI-G4-DS, where DS stands for
dense shell) show relatively weak effects or even no effect at all. However,
OS dendrimers are still much less toxic than their parental PPI-G4, especially
to peripheral blood mononuclear cells. One of our recent studies presents the
influence of non-modified (PPI-G4) and fully maltotriose modified (PPI-G4-
DS-Mal-III) generation 4 PPI dendrimers on CLL cells [174]. Both tested
dendrimers demonstrated higher cytotoxicity to CLL cells than to healthy
donor cells, whereas unmodified dendrimers were more hematotoxic. Another
study based on gene expression profiling (GEP) by microarrays identified a
New and Repositioning Approach to Treatment 43

signature associating the treatment of CLL with an open shell glycodendrimer


and FA (unpublished data). The differential analysis of the gene signature
between cultures incubated with PPI-G4-OS-Mal-III dendrimers and FA
indicated higher expression of TCF7 and BAX (unpublished data). The PPI-
G4-OS-M3 dendrimer demonstrates the ability to inhibit the increases in
proliferation with the rise in dendrimer concentration. Microarray data analysis
identified 7 out of 100 members of WNT genes (WNT10A, SFRP2, DACT1,
CDH1, WNT6, LRP5 and LDLR) whose expression was significant. Our
results reveal that glycodendrimer and FA treatment has a significant influence
on the expression of some Wnt/-catenin pathway genes in CLL (in vitro
study). The downregulation of WNT10A, WNT6 or SFRP2 expression results
in lower phosphorylation and proteosomal degradation of -catenin. LRP5 and
LDLR gene expression was also weak resulting in the blockage of the reaction
cascade and suppression of the transcription process. The loss of WNT signals
by dendrimers and FA treatment induces a reduction in the proliferation and
survival of the treatment-resistant cell line MEC-1, composed of CLL cells
with del(17p) [175]. Our recent studies have also revealed that our PPI
glycodendrimers also influence the BCR, TNF and NF-B signaling pathways,
which is very promising for targeted therapy in CLL.

CONCLUSION
Novel targeted therapies strongly suggest that the standard treatment
paradigm in CLL will change in the next few years. Particular attention should
be paid to BCR-, PI3K-, SYK-, LYN- and mTOR-targeting agents, as well as
BCL2-inhibitors, which already demonstrate encouraging activity both as
single agents and in combination with conventional chemotherapy across a
variety of B-cell neoplastic conditions, including unfavorable genetic features.
Meanwhile, as this process is very dynamic, it is without a doubt that the FDA
will approve new drugs and grant. As additional therapies are discovered and
optimized, the superiority of nanomedicines over current treatment options and
free drugs will continue to increase the efficiency of eradicating drug-resistant
cancers such as CLL. In addition, re-examining existing drugs as potential
complements for these therapies may identify very effective additive modes of
treatment. Novel and repositioning therapies continue to emerge in the
preclinical setting and will expand the armamentarium of drug combinations
for use in hematologic oncology in the coming decades.
44 Ida Franiak-Pietryga and Maria Bryszewska

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Meeting, p. 3699.

BIOGRAPHICAL SKETCHES
Name: Ida Franiak-Pietryga, Ph.D.

Summary

Over fifteen years of hands-on laboratory experience in biology and


biophysics. Assistant Professor at the Medical University in Lodz (Poland) and
a senior staff member at Lodz Copernicus Memorial Hospital (Poland).
Strong academic background from leading institutions in Europe. Trained in
hematology and genetics. Very familiar with molecular biologybased
diagnostics of leukemia. Conceived and led the research project entitiled
Dendrimers novel drugs for chronic lymphocytic leukemia (CLL). Filed
two three patents covering the usage of glicodendrimers to cure CLL. The
invention was awarded a golden medal at the 112th International Invention
Fairs Concours Lepine in 2013 in Paris. Author of numerous publications.
Coordinated complex interdisciplinary studies carried out in various institution
and countries. Creative, hard-working and focused on translational research.

Affiliation
Department of Clinical and Laboratory Genetics,
Medical University of Lodz, Poland

Address: 251 Pomorska Str, 92-213 Lodz, Poland


62 Ida Franiak-Pietryga and Maria Bryszewska

Education

1999 M.S. Laboratory Medicine,


Medical University in Lodz, Poland
2003 Ph.D. Biology, specialization in biophysics,
Faculty of Biology and Earth Sciences, University of Lodz, Poland
2012 Post-grad training in Laboratory Medical Genetics,
Medical University of Lodz, Poland

Professional experience:

Medical University in Lodz (Poland), Pharmaceutical Biochemistry


Department
11/2007-11/2010: Assistant Professor

Main accomplishments

Project leader for Assessment of leukemic cells apoptosis


mechanisms in patients with chronic lymphocytic leukemia treated
with cladribine combined with rituximab and other anti-leukemic
drugs.
Project resulted in 5 original papers, one review paper in a Polish
journal and a book chapter in English about the use of microarray
techniques in determining gene expression profiles in chronic
lymphocytic leukemia in reference to prognostic and predictive
factors. The best introduction to personalized therapy in CLL.

Laboratory of Clinical and Transplant Immunology and Genetics,


Copernicus Memorial Hospital of Lodz, Poland
1/2012 present: Specialist in oncohematological genetics

Main responsibilities and accomplishments


Directs leukemia diagnostics using mainly molecular biology
methods.
Introduced new methods and protocols to medical genetics
laboratories
New and Repositioning Approach to Treatment 63

Medical University in Lodz (Poland), Laboratory and Clinical Genetics


Department
12/2014 present: Assistant Professor

Main responsibilities
Continuation of nanoparticle research Dendrimers as a potential new
drug for CLL; pre-clinical phase, in vivo studies.
Continuation of studies of CLL cells collected from patients before
and during therapy and after relapse from therapy to determine the
genetic and biologic features associated with tumor progression,
therapeutic response and/or resistance to therapy.
Discovery of features that distinguish CLL from their normal cell
counterparts to identify new targets for therapy and/or define
surrogate markers associated with more rapid rates of cancer
progression or resistance to standard therapies.

Publications Last 3 Years:

1. Franiak-Pietryga I, Maciejewski H, Ostrowska K, Appelhans D, Voit B,


Misiewicz M, Kowalczyk P, Bryszewska M, Borowiec M. Dendrimer-based
nanoparticles for potential personalized therapy in chronic lymphocytic
leukemia: targeting the BCR-Signaling Pathway. Int J Biol Macromol 2016;
doi:10.1016/j.ijbiomac.2016.03.021 (in press) (IF=2.858)
2. Franiak-Pietryga I, Tymoniuk B, Borowiec M, Ziolkowska E, Blonski
ZJ, Janus A, Misiewicz M, Wawrzyniak E, Trelinski Jacek, Robak T,
Maciejewski H, Tomasiuk R, Cebula-Obrzut B, Smolewski P, Kaminska J,
Stepien A, Mirowski M, Gora-Tybor J. European Scientific Journal 2015,
vol.11, no. 27. ISSN: 1857-7881 (Print) e-ISSN 1857-7431.
3. Ziemba B, Franiak-Pietryga I, Pion M, Appelhans D, Muoz-
Fernndez M, Voit B, Bryszewska M, Klajnert-Maculewicz B. Toxicity and
proapoptotic activity of poly(propylene imine) glycodendrimers in vitro:
considering their contrary potential as biocompatible entity and drug molecule
in cancer. Int J Pharmaceutics 2014; 461/1-2: 391-402. DOI:
10.1016/j.ijpharm.2013.12.011 (IF= 3.458);
4. Franiak-Pietryga I, Zikowska E, Ziemba B, Appelhans D, Voit B,
Szewczyk M, Gra-Tybor J, Robak T, Klajnert B, Bryszewska M. The
influence of maltotriose-modified poly(propylene imine) dendrimers on the
chroni lymphocytic leukemia cells in vitro: dense shell G4 PPI. Molecular
Pharmaceutics 2013; 10: 2490-2501 (IF=4.782);
64 Ida Franiak-Pietryga and Maria Bryszewska

5. Rogaliska M, Franiak-Pietryga I, Boski JZ, Gralski P,


Maciejewski H, Janus A, Wawrzyniak E, Robak P, Mirowski M, Piekarski H,
Robak T, Kiliaska ZM. Towards personalized therapy for chronic
lymphocytic leukemia; DSC and cDNA microarray assessment of two cases.
Cancer Biol Ther. 2013 Jan 1;14(1):6-12. doi: 10.4161/cbt.22623. (IF= 2.636);
( equal contribution)

Research projects:

1. PBZ/MNiSW/07/2006/28 POL-POSTDOC III (2007-2010) Medical


University in Lodz project leader
Project title: Assesment of leukemic cells apoptosis mechanisms in
patients with chronic lymphocytic leukemia treated with cladribine
combined with rituximab and other antileukemic drugs.

2. A project funded by pharmaceutical company MUNDIPHARMA


concerning research on the application of a new drug in chronic
lymphocytic leukemia (2008-2010). project co-executor

3. 2011/01/B/NZ5/01371 project granted by National Science Centre


Faculty of Biology and Environmental Protection, University of Lodz,
Poland the author and the main executor of the project
Project title: Dendrimers potential drugs in chronic lymphocytic
leukemia
Project manager: Prof. Maria Bryszewska

Membership in Scientific Councils and Associations Advocating Science

1. Polish Association of Hematologists and Transfusiologists

2. Polish Association of Human Genetics

3. National Chamber of Laboratory Diagnosticians

4. European Research Initiative on CLL (ERIC)

5. US-Polish Trade Council

6. Fulbright

7. The Association of TOP500 Innovators in Poland


New and Repositioning Approach to Treatment 65

Patents:
P.401934 -- The application 4th generation poly(propylene imine)
dendrimer maltotriose modified PPI-G4-DS-Mal-III; [WIPO ST 10/C
PL401934], PCT/PL2013/000154
P.401936 -- The application 4th generation poly (propylene imine)
dendrimer maltotriose modified PPI-G4-OS-Mal-III; [WIPO ST 10/C
PL401936], PCT/PL2013/000163
P.416636 Medical application of 4th generation poly(propylene imine)
dendrimer maltotriose modified

Honors:
TOP500 Innovators in Poland (2015)
Fulbright Senior Award (2016)

Name: Maria Bryszewska, Professor

Affiliation: Department of General Biophysics,


Faculty of Biology and Environmental Protection,
University of Lodz, Poland

Education:
- University of Lodz, Faculty of Mathematics, Physics and Chemistry,
M.Sc. in physics, 1973
- University of Lodz, Faculty of Biology and Earth Sciences,
Ph.D. in biophysics, 1982
- Postdoctoral position at the Department of Biochemistry,
McMaster University, Hamilton, Canada, 1987/1988 (15 months)
- University of Lodz, Faculty of Biology and Environmental Protection,
Habilitation in biophysics (D.Sc.), 1990
- University of Lodz, Professor title in biological sciences, 1996
Address: 141/143 Pomorska St., 90-236 Lodz, Poland

Research and Professional Experience:

Major research interests


66 Ida Franiak-Pietryga and Maria Bryszewska

1) Interactions between different types of dendrimers (PAMAM, PPI,


sugar modified PPI, polylysine, viologen, carbosilane, phosphorus)
with nucleic acids, proteins, biological and model membranes.
2) Applications of dendrimers in medicine, esp. as carriers of anticancer
drugs, and antiamyloid agents in neurodegenerative disorders.
3) Studies on different types of dendrimers as delivery systems for
siRNA (siP24, siGAG1) against HIV and anti-HIV peptides.
4) Characterization of dendriplexes (dendrimers/siRNA or
dendrimers/peptides complexes) their morphology, size, surface
charge and stability (depending on time, pH, the presence of
proteolytic enzymes and other proteins).
5) Influence of dendrimers and dendriplexes on transfection and gene
silencing.
6) Toxicity studies of dendrimers and dendriplexes (cytotoxicity against
various cell lines and toxicity in vivo).
7) Genotoxicity studies - checking main cell parameters and DNA
damage upon dendrimers and whether dendrimers cause apoptosis.
8) Biological properties of mesoporous silica.

Research projects

1) H2020-TWINN-2015 VACTRAIN, Twinning on DNA-based


cancer vaccines, 2016-2018 (WP Leader)
2) Marie Curie PEOPLE-2012-IRSES NANOGENE EU-Belarus-
Russia Network In Nanomaterials-Driven Anti-Cancer Gene Therapy,
2013 2016 (co-ordinator)
3) COST Action TD0802 Biomedical applications of dendrimers 2010
2013 (co-ordinator)
4) ERA-NET EuroNanoMed Peptides associated dendrimers in
dendritic cells for the development of new nano-HIV vaccines, 2010-
2013 (WP Leader)
5) MNT ERA-NET Anti-HIV short nucleic acids transported by
nanovehicles based on dendrimers as novel therapeutical approach for
HIV-1 infection, 2009 - 2012 (WP Leader)
6) Polish National Science Centre grant Dendrimers potential drugs in
chronic lymphocytic leukemia, 2011 2014, (co-ordinator)
7) Polish National Science Centre grant Mechanisms of interactions
between dendrimers and proteins, 2012 2015, (co-ordinator)
New and Repositioning Approach to Treatment 67

Professional Appointments:
1998 till present Full Professor, University of Lodz,
Faculty of Biology and Environmental Protection,
Department of General Biophysics, Head of the Department
1990 - 1998 Associate Professor,
University of Lodz, Department of General Biophysics
1982 1990 Assistant Professor,
University of Lodz, Department of Thermobiology
1980 1982 Assistant, University of Lodz,
Department of Biophysics
1973 1980 Assistant, Medical Academy of Lodz,
Department of Biophysics

Honors:

A golden medal at the 112th International Invention Fairs Concours


Lepine in 2013 in Paris.
P.401934 -- The application 4th generation poly(propylene imine)
dendrimer maltotriose modified PPI-G4-DS-Mal-III; [WIPO ST 10/C
PL401934], PCT/PL2013/000154
P.401936 -- The application 4th generation poly (propylene imine)
dendrimer maltotriose modified PPI-G4-OS-Mal-III; [WIPO ST 10/C
PL401936], PCT/PL2013/000163
P.416636 Medical application of 4th generation poly(propylene imine)
dendrimer maltotriose modified

Publications Last 3 Years (2013-2015): H-index - 35

1) M. Neelov, A. Janaszewska, B. Klajnert, M. Bryszewska, N. Z.


Makova, D. Hicks, H. A. Pearson, G. P. Vlasov, M. Y. Ilyash, D. S.
Vasilev, N. M. Dubrovskaya, N. L. Tumanova, I. A. Zhuravin, A. J.
Turner, N. N. Nalivaeva: Molecular properties of lysine dendrimers
and their interactions with A-peptides and neuronal cells. Curr. Med.
Chem. 20(1), 134-143, 2013.
2) M. Ionov, K. Ciepluch, B. Klajnert, S. Gliska, R. Gomez-Ramirez,
F.J. de la Mata, M.A. Munoz-Fernandez, M. Bryszewska:
Complexation of HIV derived peptides with carbosilane dendrimers.
Coll. Surf. B: Biointerfaces 101, 236-242, 2013.
68 Ida Franiak-Pietryga and Maria Bryszewska

3) K. Ciepluch, M. Weber, N. Katir, A-M. Caminade, A. El Kadib, B.


Klajnert, J-P. Majoral, M. Bryszewska: Effect of viologen-phosphorus
dendrimers on acetylcholinesterase and butyrylcholinesterase
actitvities. Int. J. Biol. Macromolecules 54, 119-124, 2013.
4) K. Milowska, J. Grochowina, N. Katir, A. El Kadib, J-P. Majoral, M.
Bryszewska, T. Gabryelak: Interaction between viologen-phosphorus
dendrimers and -synuclein. J. Luminescence 134, 132-137, 2013.
5) A. Szulc, D. Appelhans, B. Voit, M. Bryszewska, B. Klajnert:
Studying complexes between PPI dendrimers and MANT-ATP. J.
Fluorescence 23, 349-356, 2013.
6) M. Ferenc, E. Pedziwiatr-Werbicka, K.E. Nowak, B. Klajnert, J-P.
Majoral, M. Bryszewska: Phosphorus dendrimers as carriers of
siRNA Characterisation of dendriplexes. Molecules 18, 4451-4466,
2013.
7) E. Vacas Cordoba, M. Pion, B. Rasines, D. Fillipini, H. Komber, M.
Ionov, M. Bryszewska, D. Appelhans, MA. Munoz-Fernadez:
Glycodendrimers as new tools in the search for effective anti-HIV
DC-based immunotherapies. Nanomedicine: Nanotechnology,
Biology and Medicine (2013), DOI:10.1016/j.nano.2013.03.004.
8) M. Ionov, K. Ciepluch, B.R. Moreno, D. Appelhans, J. Sanchez-
Nieves, R. Gomez, F.J. de la Mata, M.A. Munoz-Fernandez, M.
Bryszewska: Biophysical characterization of glycodendrimers as
nano-carriers for HIV peptides. Curr. Med. Chem. 20, 3935-3943,
2013.
9) Halets, D. Shcharbin, B. Klajnert, M. Bryszewska: Contribution of
hydrophobicity, DNA and proteins to the cytotoxicity of cationic
PAMAM dendrimers. Int. J. Pharm. 454, 1-3, 2013.
10) Franiak-Pietryga, E. Zikowska, B. Ziemba, D. Appelhans, B. Voit,
M. Szewczyk, J. Gra-Tybor, T. Robak, B. Klajnert, M. Bryszewska:
The influence of maltotriose-modified poly(propylene imine)
dendrimers on the chronic lymphocytic leukemia cells in vitro: dense
shell G4 PPI. Mol. Pharm. 10, 2490-2501, 2013.
11) E. Pedziwiatr-Werbicka, E. Fuentes, V. Dzmitruk, J. Sanchez-Nieves,
M. Sudas, E. Drozd, A. Shakbazau, D. Shcharbin, F.J. de la Mata, R.
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C carbosilane dendrimers as carriers for anti-HIV nucleic acids:
Studies on complexation and interaction with blood cells. Coll. Surf.
B: Biointerfaces 109, 183-189, 2013.
New and Repositioning Approach to Treatment 69

12) N. Borisevich, S. Loznikova, A. Sukhodola, I. Halets, M. Bryszewska,


D. Shcharbin: Acidosis, magnesium and acetylsalicylic acid: Effects
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Spectroscopy 104, 158-164, 2013.
13) N. Shcharbina, D. Shcharbin, M. Bryszewska: Nanomaterials in
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14) Lazniewska, K. Milowska, N. Katir, A. El Kadib, M. Bryszewska, J.P.
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of cationic phosphorus dendrimer toxicity against murine neural cell
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70 Ida Franiak-Pietryga and Maria Bryszewska

22) A. Janaszewska, M. Ciolkowski, D. Wrbel, J.F. Petersen, M. Ficker,


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New and Repositioning Approach to Treatment 71

31) D. Shcharbin, A. Janaszewska, B. Klajnert-Maculewicz, B. Ziemba,


V. Dzmitruk, I. Halets, S. Loznikova, N. Shcharbina, K. Milowska,
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33) V. Potkin, D. Shcharbin, A.A. Denisov, S.G. Paschkevich, M.
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34) A. Shakhbazau, S.J. Archibald, D. Shcharbin, M. Bryszewska, R.
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35) E. Pdziwiatr-Werbicka, K. Miowska, M. Podlas, M. Marcinkowska,
M. Ferenc, Y. Brahmi, N. Katir, J-P. Majoral, A. Felczak, A.
Boruszewska, K. Lisowska, M. Bryszewska, A. El Kadib:
Oleochemical-tethered SBA-15-type silicates with tunable nanoscopic
order, carboxylic surface, and hydrophobic framework: cellular
toxicity, hemolysis, and antibacterial activity. Chem. Eur. J. 20, 1-12,
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36) D. Shcharbin, N. Shcharbina, M. Bryszewska: Recent patents in
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37) Wroska, A. Felczak, K. Zawadzka,A. Janaszewska, B. Klajnert, M.
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ciprofloxacin. New. J. Chem. 38, 2987-2992, 2014.
38) D. Shcharbin, N. Shcharbina, K. Milowska, F.J. de la Mata, M.A.
Munoz-Fernandez, S. Mignani, R. Gomez-Ramirez, J-P. Majoral, M.
Bryszewska: Interference of cationic polymeric nanoparticles with
clinical chemistry tests Clinical relevance. Int. J. Pharm. 473, 599-
606, 2014.
72 Ida Franiak-Pietryga and Maria Bryszewska

39) K. Milowska, A. Szwed, M. Zablocka, A-M. Caminade, J-P. Majoral,


S. Mignani, T. Gabryelak, M. Bryszewska: In vitro PAMAM,
phosphorus, and violo gen-phosphorus dendrimers prezent rotenone-
induced cell damage. Int. J. Pharm. 474, 42-49, 2014.
40) Sz. Sekowski, M. Ionov, M. Kaszuba, S. Mavlyanov, M. Bryszewska,
M. Zamaraeva: Biophysical studies on interaction between
hydrolysable tannins isolated from Oenothera gigas and Geranium
sanguineum with human serum albumin. Coll. Surf. B: Biointerfaces
123, 623-628, 2014.
41) A. Szulc, M. Zablocka, Y. Coppel, Ch. Bijani, W. Dabkowski, M.
Bryszewska, B. Klajnert-Maculewicz, J-P. Majoral: A viologen-
phosphorus dendritic molecule as a carrier of ATP and Mant-ATP:
spectrofluorimetric and NMR studies. New J. Chem. 38, 6212-6222,
2014.
42) Mignani S., Bryszewska M., Klajnert-Maculewicz B., Zablocka M.,
Majoral J. P., Advances in combination therapies based on
nanoparticles for efficacious cancer treatment: An analytical report.
Biomacromolecules 16, 1-27, 2015.
43) Ionov M., Ciepluch K., Garaiova Z., Melikishvili S., Michlewska S.,
Gliska S., Balcerzak ., Miowska K., Shcharbin D., Gomez-
Ramirez R., de la Mata F.J., Waczulikova I., Bryszewska M., Hianik
T., Dendrimers complexed with HIV-1 peptides interact with
liposomes and lipid monolayers. Biochim. Biophys. Acta
Biomembranes, 1848, 907- 915, 2015.
44) M. Ferenc, N. Katir, K. Miowska, M. Bousmina, J-P. Majoral, M.
Bryszewska, A. El Kadib, Haemolytic activity and cellular toxicity of
SBA-15-type silicas: Elucidating the role of the mesostrucrure, the
surface functionality and the linker length. J. Mater. Chem. B 3, 2714-
2724, 2015.
45) Nowacka, K. Milowska, M. Bryszewska: Interaction of PAMAM
dendrimers with bovine insulin depends on nanoparticle end-groups.
J. Lumin. 162, 87-91, 2015.
46) D. Shcharbin, N. Shcharbina, A, Shakhbazau, S. Mignani, J.P.
Majoral, M. Bryszewska: Phosphorus-containing nanoparticles:
biomedical patents review. Expert Opin. Ther. Patents 25(5), 539-548,
2015
47) S. Moreno, A. SzwedN. El Brahmi, K. Milowska, J. Kurowska, E.
Fuentes-Paniagua, E. Pedziwiatr-Werbicka, T. Gabryelak, N. Katir,
F.J. de la Mata, Ma.A. Munoz-Fernandez, R. Gomez-Ramirez, A.M.
New and Repositioning Approach to Treatment 73

Caminade, J.P. Majoral, M. Bryszewska: Synthesis, characterization


and biological properties of new hybrid carbosilane-viologen-
phosphorus dendrimers. RSC Adv. 5, 25942-25958, 2015.
48) K. Milowska, A. Szwed, M. Mutrynowska, R. Gomez-Ramirez, F.J.
de la Mata, T. Gabryelak, M. Bryszewska: Carbosilane dendrimers
inhibit -synuclein fibrillation and prezent cells from rotenone-
induced damage. Int. J. Pharm. 484, 268-275, 2015.
49) Ionov, J. Lazniewska, V. Dzmitruk, I. Halets, S. Loznikova, D.
Novopashina, E. Apartsin, O. Ktasheninina, A. Venyaminova, K.
Milowska, O. Nowacka, R. Gomez-Ramirez, F.J. de la Mata, J-P.
Majoral, D. Shcharbin, M. Bryszewska: Anticancer siRNA cocktails
as a novel tool to treat cancer cells. Part (A). Mechanisms of
interaction. In. J. Pharm. 485, 261-269, 2015.
50) V. Dzmitruk, A. Szulc, D. Shcharbin, A. Janaszewska, N. Shcharbina,
J. Lazniewska, D. Novopashina, M. Buyanova, M. Ionov, B. Klajnert-
Maculewicz, R. Gomez-Ramirez, S. Mignani, J-P. Majoral, M.A.
Munoz-Fernandez, M. Bryszewska: Anticancer siRNA cocktails as a
novel tool to treat cancer cells. Part (B). Efficiency of
pharmacological action. Int. J. Pharm. 485, 288-294, 2015.
51) D. Shcharbin, M. Ionov, V. Abashkin, S. Loznikova, V. Dzmitruk, N.
Shcharbina, L. Matusevich, K. Milowska, K. Gacki, S. Wysocki, M.
Bryszewska: Nanoparticle corona for proteins: mechanisms of
interaction between dendrimers and proteins. Coll. Surf. B:
Biointerfaces 134, 377-383, 2015.
52) K. Milowska, A. Rybczyska, J. Mosiolek,J. Durdyn, E.M. Szewczyk,
N. Katir, Y. Brahmi, J-P. Majoral, M. Bousmina, M. Bryszewska, A.
El Kadib: Biological activity of mesoporous dendrimer-coated
titanium dioxide: insight on the role of the surface-interface
composition and the framework crystallinity. ACS Appl. Mater.
Interfaces 7, 19994-20003, 2015.
53) A. Shakhbazau, M. Mishra, T-H. Chu, C. Brideau, K. Cummins, S.
Tsutsui, D. Shcharbin, J-P. Majoral, S. Mignani, M. Blanchard-Desce,
M. Bryszewska, V. Wee Yong, P.K. Stys, J. van Minnen: Fluorescent
phosphorus dendrimer as a spectral nanosensor for macrophage
polarization and fate tracking in spinal cord injury. Macromol. Biosci.
15(11), 1523-1534, 2015.
54) Wroska, A. Felczak, K. Zawadzka, M. Poszepczyska, S. Ralska,
M. Bryszewska, D. Appelhans, K. Lisowska: Poly(propylene imine)
74 Ida Franiak-Pietryga and Maria Bryszewska

dendrimers and amoxicillin as dual-action antibacterial agents.


Molecules 20, 19330-19342, 2015.
55) K. Ciepluch, B. Nystrm, D. Appelhans, M. Zablocka, M.
Bryszewska, J-P. Majoral: Effect of dendritic polymers on a Simple
model biological membrane. J. Polymer Res. 22, 2015, DOI:
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56) D. Wrobel, A. Janaszewska, D. Appelhans, B. Voit, M. Bryszewska,
J. Maly: Interactions of dendritic glycopolymer with erythrocytes, red
blood cell ghosts and membrane enzymes. Int. J. Pharm. 496, 475-
488, 2015.
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Mignani, M. Zablocka, J-P. Majoral, M. Bryszewska, B. Klajnert-
Maculewicz, J. Cladera: Fourier trans form infrared spectroscopy
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Chapters in books

1) R.M. Epand, M. Bryszewska: Glucagon and Insulin Receptor. In:


Comprehensive Medicinal Chemistry. Vol. 3, Pergamon Press, 1989.
2) B. Klajnert, M. Bryszewska: Dendrimers as delivery systems in gene
therapy. In: New Developments in Mutation Research, pp. 217-240,
Nova Sci. Publ., Inc., Ed. Ch. Valon, 2007, ISBN 1-59454-664-9.
3) B. Klajnert, M. Bryszewska: Affinity of dendrimers for proteins
and peptides biomedical implications. In: Nanotechnological
Applications of Novel Polymers. Transworld Research Network, Ed.
Mohsen Adeli, pp. 51-71, 2009, ISBN 978-81-7895-412-7.
4) M. Bryszewska, B. Klajnert: Dendrimers as drug carriers. In:
Nanoparticles: Synthesis, Characterization and Applications. Ed. R.S.
New and Repositioning Approach to Treatment 75

Chaughule and R.V. Ramanujan, pp. 345-357, ISBN 1-58883-180-9,


2010.
5) B. Klajnert, M. Bryszewska, Dendrimeric polymers for pharma
applications anti-cancer therapies. rozdzia w Nano-architectures
for solubilization and delivery in food, cosmetic and pharma
applications (Eds. N. Garti, I. Yuli-Amar) DEStech Publications, Inc.
Lancaster, PA USA, 363-377, 2011.
6) V. Dzmitruk, D. Shcharbin, E. Pedziwiatr-Werbicka, M. Bryszewska.
Dendrimers in Anti- HIV Therapy. In: Advances in Nanocomposite
Technology, Ed. A. Hashim, InTech, Croatia, ISBN 978-953-307-
347-7, 2011.
7) R. Gomez, F.J. de la Mata, J.L. Jimenez-Fuentes, P. Ortega, B.
Klajnert, E. Pedziwiatr-Werbicka, D. Shcharbin, M. Bryszewska, M.
Maly, J. Maly, M.J. Serramia, R. Lorente, M.A. Munoz-Fernandez:
Cationic carbosilane dendrimers as non-viral vectors of nucleic acids
(oligonucleotide or siRNA) for gene therapy purposes. In: Dendrimers
in Biomedical Applications. Eds. B. Klajnert, Ling Peng and Valentin
Cena, RSC Publishing, pp. 40-55, 2013, ISBN:978-1-84973-611-4.
8) D. Appelhans, N. Benseny, O. Klementieva, M. Bryszewska, B.
Klajnert, J. Cladera: Dendrimers as antiamyloidogenic agents.
Dendrimer-amyloid aggregates morphology and cell toxicity. In:
Dendrimers in Biomedical Applications. Eds. B. Klajnert, Ling Peng
and Valentin Cena, RSC Publishing, 2013, pp. 1-13, ISBN:978-1-
84973-611-4.
9) K. Gardikis, E.A. Mourelatou, M. Ionov, A. Aserin, D. Libster, B.
Klajnert, M. Bryszewska, N. Garti, J.P. Majoral, K. Dimas, C.
Demetzos: Natural and synthetic biomaterials as composites of
advanced drug delivery nanosystems (ADDNSS). Biomedical
applications. Ibid, pp. 30-39.
10) M.F. Ottaviani, D. Appelhans, F.J. de la Mata, S. Garcia-Gallego, R.
Mazzeo, M. Cangiotti, L. Fiorani, J.P. Majoral, A.M. Caminade, M.
Bryszewska, B. Klajnert: Characterization of dendrimers and their
interactions with biomolecules for medical use by means of electron
magnetic resonance. Ibid, pp. 115-133.
11) P. Posocco, E. Laurini, V. Dal Col, D. Marson, L. Peng, D.K. Smith,
B. Klajnert, M. Bryszewska, A-M. Caminade, J.P. Majoral, M.
Fermeglia, K. Karatasos, S. Pricl: Multiscale modeling of dendrimers
and dendrons for drug and nucleic acid delivery. Ibid, pp. 148-166.
76 Ida Franiak-Pietryga and Maria Bryszewska

Books

1) Barbara Klajnert, Maria Bryszewska: Dendrimers in Medicine. Nova


Sci. Publ., Hauppauge, USA, 2007, ISBN: 1-60021-664-1.
2) D. Shcharbin, B. Klajnert, M. Bryszewska, Practical Guide to
Studying Dendrimers. iSmithers Rapra Publishing, 2010 ISBN: 978-
1-84735-444-0.
In: Chronic Lymphocytic Leukemia ISBN: 978-1-63485-510-5
Editor: Kimberly Rodriquez 2016 Nova Science Publishers, Inc.

Chapter 3

PROGNOSTIC AND PREDICTIVE INDICATORS


IN CHRONIC LYMPHOCYTIC LEUKEMIA

Nili Saar1, Pia Raanani1,2 and Uri Rozovski1,2,*


1
Division of Hematology, Davidoff Cancer Center,
Rabin Medical Center, Petah-Tikva, Israel
2
Sackler Faculty of Medicine, Tel Aviv University,
Tel Aviv, Israel

ABSTRACT
Several prognostic factors are available for patients with CLL who
require therapy. Of these, high levels of 2 microglobulin, adverse
cytogenetics and high expression levels of CD38 and ZAP70 surface
markers are well described and mark patients with unfavorable
prognosis [1]. In approximately 50% of patients, more than 2% of the
immunoglobulin variable heavy-chain gene (IGHV) nucleotide sequence
differs from that of germ-line non-clonal DNA [2]. Patients with mutated
IGHV respond better to chemo-immunotherapy [3], and patients with
unmutated IGHV have a shorter time to treatment, shorter time to next
treatment, inferior response to chemotherapy, higher rates of
chemotherapy resistance, and lower survival rates [4-6].
Until recently, CLL was considered an incurable disease. However,
two recent studies found that with the combination of fludarabine,

*
Corresponding Author: Uri Rozovski, MD; Institute of Hematology; Davidoff Cancer Center,
Beilinson Hospital; Petah-Tikva, Israel, 49100; Email: rozovski.uri@gmail.com.
78 Nili Saar, Pia Raanani and Uri Rozovski

cyclophosphamide and rituximab (FCR) a fraction of patients with CLL


achieve long-term disease-free survival and are probably cured [7, 8].
Because none of the novel agents that have been approved or are
currently being tested cures CLL, it has been argued that chemo-
immunotherapy will remain the treatment of choice, in particular for
patients with mutated IGHV in the foreseen future.
In this chapter we will review the various prognostic markers that
help clinicians predict the clinical course and response to therapy of
treatment nave CLL patients. We will highlight IGHV mutation status as
a predictive marker that could assist in selecting treatment from the
growing arsenal of available drugs for CLL.

HISTORICAL PERSPECTIVE
Chronic lymphocytic leukemia (CLL) is characterized by the gradual
accumulation of functionally incompetent, mature appearing lymphocytes, co-
expressing B-lymphocyte antigens (CD19, CD23) and aberrantly expressing
CD5 [9]. That the natural history of CLL is extremely variable was recognized
long ago. In the first case series published in 1924, Minot and Isaac reported
that approximately 30% of patients with CLL were long-term survivors.
However, most patients died few months to 5 years from diagnosis, usually
from CLL-related conditions [10] [Figure 1]. Staging systems developed
during the 1970's [11, 12] are still used today to delineate patients who require
therapy from those with indolent CLL, who are followed without treatment
[13]. In patients who require therapy, selection of first-line treatment is mainly
based on patient-related factors [13-15]. Disease-related factors are commonly
used to predict response to therapy and overall outcome, and only occasionally
to select a first line treatment modality [16-18]. In patients with relapsed
disease, the extent and duration of response to previous treatment are taken
into account, and specific abnormalities such as del(17p) or TP53 mutation
may guide therapy selection [13, 14].
Prognostic and Predictive Indicators in Chronic Lymphocytic 79

Adapted with permission from Figure 3 in Minot and Isaacs (1924) Chronic Lymphatic
Leukemia: Age Incidence. Duration and Benefit Derived from Irradiation, The
Boston Medical Journal, 191(1) @ The Massachusetts Medical Society.

Figure 1. First case series of patients with CLL.

In an era of a growing arsenal of therapeutic modalities on the one hand,


and the likelihood of cure with standard therapy for certain subgroups on the
other, the significance of prognostic and predictive markers in CLL is
enormous. In this chapter we will review the various prognostic tools (markers
and scoring systems) employed in current practice and highlight their role in
predicting response to therapy. A special consideration will be given to the
assay that detects mutations in the immunoglobulin heavy chain genes as a
reliable predictor of cure in patients treated with chemo-immunotherapy.

TRADITIONAL STAGING SYSTEMS


The Rai [11] and Binnet [19] staging systems are the most commonly used
staging systems in CLL [13, 14]. A staging system that integrates both
methods was proposed by the International workshop on CLL (IWCLL) in
1981, but was not widely accepted. These staging systems stratify newly
diagnosed CLL patients into 3 (Binnet or revised Rai [20]) stages and their
80 Nili Saar, Pia Raanani and Uri Rozovski

most important outcome is that patients with early-stage disease need not be
treated [13, 14].
The Rai system is based on hierarchical grouping of disease
manifestations and assumes a gradual increase in the burden of leukemic
lymphocytes, starting in the blood and bone marrow (lymphocytosis),
progressively involving lymph nodes (lymphadenopathy), spleen and liver
(organomegaly), and eventually compromising bone marrow function (anemia
and/or thrombocytopenia) [11]. The original Rai system included 5 stages.
However, as there were only 3 actuarial survival curves, it was modified to
include 3 stages instead, corresponding with low, intermediate and high risk
features [20].
The Binnet system, commonly used in Europe, takes five potential sites
of involvement into consideration: cervical, axillary, and inguinal lymph
nodes, spleen, and liver. Patients are classified according to the number of
involved sites, plus the presence of anemia (hemoglobin <10 g/dL) and/or
thrombocytopenia (platelets <100,000/microL).

CYTOGENETIC ABNORMALITIES
Cytogenetic abnormalities are detected by fluorescence in situ
hybridization (FISH) in approximately 80% of patients with CLL, and are
independent predictors of disease progression and survival [21]. The most
commonly used panel in clinical practice includes del(13q), trisomy 12,
del(11q), and del(17p). The 'International Workshop for CLL' guidelines call
for FISH testing of all newly diagnosed patients using probes for 13q, T12,
11q, 6q, and 17p cytogenetic abnormalities [13], a model which was recently
validated in a large cohort of patients [22]. However, with the exception of
del(17p) testing, FISH analysis is not used for follow-up or therapy-related
decisions in current practice.
The most common genetic abnormality in CLL is deletion of 13q14,
which occurs in more than 50% of patients [23]. Individuals with 13q14
deletions have a relatively benign disease that usually manifests as stable or
slowly progressive isolated lymphocytosis. The common deleted region
includes two micro-RNA genes, miR-15a and miR-16-1 [23] that have been
shown to negatively regulate Bcl-2 expression, allowing CLL cells to evade
apoptosis [23-28].
Trisomy 12 is found in 15% of patients and confers an intermediate
prognostic risk [21, 29]. When compared to patients who were negative
Prognostic and Predictive Indicators in Chronic Lymphocytic 81

for trisomy 12, patients with trisomy 12 had higher incidence of


thrombocytopenia, Richter transformation and second malignant neoplasms
[30]. Therefore, tight post-treatment surveillance is needed in this subgroup of
patients.
Del(11q) is detected in 520% of CLL patients [21]. While highly variable
in size, the minimal deleted region in this aberration includes the ataxia
telangiectasia mutated (ATM) gene. Patients with del(11q) are diagnosed at a
younger age, tend to have bulky lymphadenopathy, shorter time to first
treatment and shorter OS [21, 31-35]. However, treatment with FCR is
particularly beneficial in these patients and may overcome the adverse
prognosis associated with this abnormality [36, 37].
Del(17p) is found in approximately 38% of CLL patients at diagnosis
[21, 38-40], but accounts for up to 50% of chromosomal aberrations in patients
with relapsed / refractory disease [38, 41]. Del(17p) patients demonstrate
shortest time from diagnosis to first treatment, lowest response rates to front-
line and salvage therapy with standard chemo-immunotherapy, and short
remission durations [21, 35, 41-45]. Aberrations involving the short arm of
chromosome 17 may affect the TP53 tumor suppressor gene. Over 90% of
patients with del(17p) have concurrent TP53 mutations [38]. TP53 mutations
may also occur in the absence of del(17p) and carry a poor prognosis
regardless of the presence of del(17p) [41]. However, even within this group,
clinical course varies and only half of patients with del(17p) developed an
indication for treatment during a 3 year observation period. Del(17p) patients
with mutated IGHV (M-CLL), who do not have complex karyotypes,
demonstrate a particularly extended time to first treatment [46, 47].
Patients harboring 17p deletions and/or TP53 mutations do not respond to
standard treatment regimens with FC (fludarabine-cyclophosphamide) or FCR
(fludarabine-cyclophosphamide-rituximab), in part due to lack of wild-type
p53 function, an important pathway for mediating the cytotoxicity of purine
analogs. In the randomized front-line FC vs. FCR GCLLSG CLL8 trial [36],
only 1 of 22 patients with del(17p) (5%) in the FCR arm achieved complete
remission, and the median progression free survival (PFS) in that arm was
only 11.3 months. Similarly, in the German front-line BR (bendamustine-
rituximab) trial, none of the 8 patients with deletion 17p achieved CR, and the
median PFS was only 7.9 months [48].
In order to improve survival of non-responding patients, a wide spectrum
of new drugs acting independently on p53 has been tested, including
flavopiridol, lenalidomide, alemtuzumab, and rituximab [49]. Recently,
inhibitors of key pathways in tumor B-cell pathophysiology have arisen as
82 Nili Saar, Pia Raanani and Uri Rozovski

promising new drugs in terms of overall response, duration of response, and


progression-free survival. Among them the Brutons tyrosine-kinase (BTK)
inhibitor ibrutinib [50, 51], the cyclin-dependent kinase (CDK) inhibitor
dinaciclib [52, 53], and the Bcl-2 inhibitor venetoclax have demonstrated
significant activity in CLL with del (17p) [54].

STIMULATED CLL KARYOTYPING


Traditional mitogens are not effective in CLL. Therefore, karyotyping of
CLL cells fails in most cases. CpG-oligodeoxynucleotides effectively
stimulate CLL cells [55-57] and provide consistent results across different
laboratories [58]. As complex karyotype is not detected by FISH, stimulated
CLL karyotyping provides additional prognostic information in CLL [59, 60].
Complex karyotype has been associated with inferior outcome in treatment
nave patients [61], but was mainly studied as a prognostic indicator in
the relapsed/refractory setting [62, 63], including in patients undergoing
hematopoietic stem cell transplantation [64]. More recently, complex
karyotype predicted inferior disease free survival (DFS) and overall survival
(OS) in patients treated with ibrutinib [65], and it has been shown that del(17p)
patients are most likely to progress on ibrutinib therapy if they have a complex
karyotype. Those without complex karyotypes appear to have equivalent
outcomes to pts. without del(17p) on ibrutinib [65].

IGHV MUTATION STATUS


In approximately half of CLL patients, the immunoglobulin variable
heavy chain gene (IGHV) exhibits more than a 2% difference in its nucleotide
sequence compared with germ-line non clonal DNA [2]. In 1999 two groups
reported independently that unmuted IGHV (U-CLL) patients experienced
shorter DFS and OS [66, 67]. Since then, IGHV mutation status has emerged
as one of the most powerful prognostic and predictive factors in CLL. Patients
with U-CLL have shorter time to first treatment, shorter time to next treatment,
and shorter DFS and OS [66-72]. Although patients with U-CLL have higher
rates of chemo-refractoriness [73] some of the novel agents have good activity
in U-CLL. Specifically, lenalidomide, a second generation immunomodulatory
drug, has similar activity in M-CLL and U-CLL [74, 75], and with ibrutinib
Prognostic and Predictive Indicators in Chronic Lymphocytic 83

treatment the overall response rate in patients with U-CLL is even higher than
in M-CLL [76]. IGHV mutation status is one of the most important prognostic
factors used to stratify patients in clinical trials [77, 78]. However, because as
many as 50% of patients with M-CLL treated with FCR are likely to get cured
[7, 8], stratifying patients by IGHV mutation status should be considered
outside clinical trials, at least in patients who are candidates for treatment with
FCR.

SERUM MARKERS
Thymidine kinase (TK) is a cell cycle-dependent enzyme of the pyrimidine
salvage pathway [79, 80]. In order to be incorporated into the DNA, the
nucleoside thymidine must undergo phosphorylation, a step catalyzed by
thymidine kinase [81]. There exist at least two TK isoenzymes, which differ in
their properties and cellular distribution. Since TK1, the cytosolic isozyme, is
synthesized only in the G1/S phase of dividing cells, and is absent in resting
cells, it has been studied as a marker of proliferation [82-85].
Serum TK1 levels have been reported to have prognostic significance in
patients with low-grade non-Hodgkins lymphomas and CLL [86-92]. In CLL,
high levels of TK1 predict early progression [91], shorter survival, and
increased risk of Richter's transformation [92]. The cumbersome radioisotope
assay used to detect TK1 in serum hampered the application of TK1 as a
prognostic marker. With the introduction of new anti-TK1 antibody-based
assays, TK levels have become more clinically relevant [79]. Still, quantifying
serum TK1 is not in widespread use in routine practice.
Beta-2 microglobulin (B2M) is ubiquitously expressed in nucleated cells
and is an extracellular component of the MHC class 1 and other class 1-like
molecules [93]. Because CLL cells constitutively shed B2M [94], serum levels
are considered a reliable marker of tumor burden [95, 96] and correlate well
with disease stage and prognosis [97, 98]. B2M was deemed valuable even in
a subset of patients with early disease: patients with Binnet stage A CLL and
low levels of B2M were less likely to require therapy during a 3.5 year follow
up [96]. High levels of B2M are an independent adverse prognostic factor for
patients treated with FCR [99], and normalization of B2M during the first 6
months of treatment is predictive of superior progression free survival (PFS)
for patients treated with ibrutinib-based regimens [100].
CD23 is a B-lymphocyte specific antigen, constitutively expressed on
CLL cells [101]. When expressed on cell surface, CD23 functions as a low
84 Nili Saar, Pia Raanani and Uri Rozovski

affinity receptor for IgE [102]. After auto-proteolytic cleavage, soluble CD23
(sCD23) is detected in serum and functions as a potent mitogenic growth
factor which provokes entry of resting (Go) B cells into the G1/S phase [103].
Detection of CD23 on cell surface helps distinguish CLL (CD5+, CD23+)
from mantle cell lymphoma (CD5+, CD23-) [104]. High sCD23 in CLL
correlates with disease activity [105] and predicts shorter time to disease
progression and a shorter overall survival [106, 107].

CELL SURFACE MARKERS


CD38 - Expressed by most cells of the hematopoietic lineage at some
point during differentiation [108], greater expression of the CD38 surface
protein is correlated with poor prognosis [68, 109-114]. CD38 expression in
CLL marks cells with proliferation capacity [115, 116], and correlates with
unmutated IGHV [66]. CD38 expression predicts shorter time to first treatment
[114], shorter DFS [110, 111, 113] and OS [68, 111, 112, 114] and
chemotherapy resistance [113, 114]. However, levels may vary with time [67],
and the threshold cutoff ranges from 0-30% in different studies [66-68, 72,
110, 112, 113]. Clearly, the most reliable method to use CD38 expression as a
prognostic marker in CLL has not been determined yet.
ZAP-70 - zeta-chain-associated protein kinase 70 (ZAP-70) is normally
expressed in T cells and natural killer cells [117]. Aberrant expression of ZAP-
70 in CLL has been associated with worse outcome [118-121]. In T-cells,
ZAP-70 is an integral part of signal transduction through the T-cell receptor, in
charge of phosphorylating tyrosine-containing immuno-receptor tyrosine-
based activation motifs (ITAMs) and activating downstream signaling
pathways [117, 122-128]. When present in CLL, ZAP-70 is engaged in B-cell
receptor signaling [129, 130].
Levels of ZAP-70 are quantified either by flow cytometry [118, 131],
immunohistochemistry [129] or by detecting the mRNA transcript by PCR
[132]. When present, ZAP-70 is associated with shorter time to first treatment
[120, 133], shorter PFS [118, 119, 134], and shorter OS [118, 119, 135]. It is
also strongly associated with U-CLL [120, 129, 135, 136].
CD49d - A part of the integrin family, the cell surface glycoprotein
CD49d facilitates cell to cell and cell to extracellular matrix attachments [137].
It is expressed in 40% of CLL cases but almost universally in trisomy 12
[138]. Since CD49d mediates the binding of CLL cells to fibronectin, it was
suggested that CD49d mediated cellular to extracellular interaction could
Prognostic and Predictive Indicators in Chronic Lymphocytic 85

explain why trisomy 12 positive cells preferentially home to lymph nodes


[139]. High expression of Cd49d is associated with shorter time to first
treatment and shorter OS in CLL [140-146].

SOMATIC MUTATIONS
Whole exome sequencing studies revealed relatively low mutation burden
[147-149], and only few recurrently mutated genes in CLL. Among 3334
patients tested for the 5/6 most frequently mutated genes in CLL, mutated
SF3B1 was detected in 11%, TP53 in 10%, NOTCH1 in 8%, and BIRC or
MYD88 were each detected in 2% of patients [150]. Mutations in ATM,
detected in a substantial minority of CLL patients across different series [151-
154], were not tested in this study. Based on their strong predictive power,
sometimes independent of other prognostic indicators, it has been suggested
that at least some of these mutations be integrated into the current hierarchical
model, based on FISH findings [155].
SF3B1: Mutated in CLL but also in myeloid malignancies, SF3B1 codes
for a protein that is part of the spliceosome, a complex of RNA and protein
subunits that takes part in alternative splicing [156], and other cellular
processes [157]. Mutations in SF3B1 confer an adverse prognosis, independent
of other prognostic markers in CLL [147, 148, 150], are more frequent in
patients with progressive disease [148, 150], and predict shorter time to first
treatment [147, 150, 158] as well as shorter PFS and OS [148, 158].
TP53: Located on the short arm of chromosome 17, the tumor suppressor
gene TP53 is the most frequently mutated gene in human cancers [159]. As a
primary gatekeeper, the p53 transcription factor will halt the cell cycle at G1
when damaged DNA is identified [160-165], and loss of p53 function will
cause genomic instability and accumulation of mutations [161]. In patients
with del(17p), mutated TP53 in the remaining allele occurs in 81-94% of
patients and predicts poor survival outcome [41, 43, 44, 166]. However,
whereas one prospective study did not find prognostic significance to the
presence of mutated TP53 in non-del(17p) CLL [44], most found that mutated
TP53 carries a poor prognosis independent of del(17p) [45, 166, 167]. Mutated
TP53 predicts resistance to standard chemo-immunotherapy [41, 43, 168-170],
early relapse [41, 168] and poor survival outcome [41, 43, 166, 168-170].
Even small mutated TP53 sub-clones found by sensitive next generation
sequencing in untreated CLL patients predict poor survival [167].
86 Nili Saar, Pia Raanani and Uri Rozovski

Some of the novel drugs that are currently available or tested in clinical
trials may be safe and effective even in patients with TP53 aberrations. For
example, overall response rate, PFS and OS were comparable in patients with
relapsed CLL and TP53 mutations treated with the oral immunomodulatory
agent lenalidomide [171]. Likewise, high response rates were observed in
treatment-nave and relapsed patients treated with the Bruton Tyrosine Kinase
(BTK) inhibitor ibrutinib [50, 172, 173]. It is therefore imperative to test for
TP53 mutations when treatment decisions are taken. The European Research
Initiative on CLL (ERIC) recommends TP53 mutation analysis in all CLL
patients who are candidates for treatment [174].
ATM: Located on the long arm of chromosome 11, the ataxia
telangiectasia-mutated (ATM) gene spans 146kd in 66 divided exons. Because
of its large size, it has been difficult to delineate polymorphisms from
pathogenic mutations, but alterations in ATM, including both mutations and
deletions, occur in 25% of patients with CLL at diagnosis [175]. Similar to
TP53, the ATM gene codes for a checkpoint inhibitor, and del11(q), detected
by FISH, is second only to del17(p) as the most unfavorable genetic
abnormality in CLL [176]. In patients with del11(q), ATM point mutations in
the second allele occur in 20% to 40% and predict worse outcome [175, 177].
NOTCH1: NOTCH1 encodes for a transmembrane protein that upon
ligand binding undergoes proteolytic cleavage, releasing an intracellular
domain which translocates to the nucleus and functions as a transcription
factor [178, 179]. In CLL, a frameshift mutation in codon 2515 results in a
truncated constitutively active intracellular protein. NOTCH1 mutations occur
more frequently with trisomy 12 [147, 180-182] and U-CLL [181-183].
Though not an independent prognostic indicator, they are associated with
advanced disease and poor prognosis [149, 181, 183-185].
BIRC3: BIRC3 encodes for a negative regulator of NF-B. Mutations in
BIRC3 lead to constitutive activation of NF-B, and have been associated with
U-CLL, advanced disease [150] and chemo-refractoriness to fludarabine-based
treatments [186].
MYD88: Mutations in this adaptor protein of the toll-like receptor-NF-B
pathway, are frequent in lymphoproliferative disorders, and the same leucine-
proline substitutions are found in lymphoplasmacytic leukemia (87% of
patients), diffuse large B-cell lymphoma (15% of patients) and CLL (3% of
patients), where it is associated with M-CLL and a favorable clinical course
[187].
Table 1. Prognostic and predictive indicators in treatment nave patients with CLL

Patient Staging Disease burden Disease Serum Cell Chromosomal IGHV Somatic
characteristics Kinetics markers surface aberrations status mutations
markers
Age 60 Rai / Binet Absolute Lymphocyte thymidine CD38 del(13q) Mutated TP53
lymphocyte doubling kinase vs. non-
Male sex count time ZAP-70 trisomy(12) mutated ATM
2-
ECOG 1 Marrow microglobulin CD49d del(11q) VH3.21 SF3B1
infiltration gene
pattern soluble CD23 del(17p) usage NOTCH1
(diffuse/non-
diffuse) del(6q) BIRC3

Computed complex MYD88


Tomography chromosomal
abberations
Table 2. Prognostic scales in treatment nave patients with CLL

Cohort (N) Scoring Outcome


MDACC 2007 [207] MD Anderson Cancer Center 1 point 5-year OS
(1674) age<50
2m 1-2xULN Low risk (1-3 points): 97%
ALC 20-50109/L Intermediate (4-7 points): 80%
male sex High ( 8 points): 55%
Rai III/IV
3 involved nodal groups
2 points
age 50-65
2m >2xULN
ALC >50109/L
3 points
age>65
MDACC [35] MD Anderson Cancer Center ** See below TTFT
(930)
GCLLSG [208] 350 European Centers (1948) 1 point 5-year OS
del(11q)
1.7 < 2m 3.5 mg/L low (0-2 points): 95%
U-CLL intermediate (3-5 points): 87%
ECOG >0 high (6-10 points): 68%
male Sex very high (11-14 points): 19%
age >60 y
Cohort (N) Scoring Outcome
2 points
TK >10.0U/L
2m >3.5 mg/L
6 points
Del(17p)
CLL-IPI [209] 8 multicenter phase 3 clinical 1 point 5-year OS
trials (3472) age >65 years
Rai I-IV / Binet B/C Low (0-1 points): 93%
2 points intermediate (2-3 points): 80%
U-CLL high (4-6 points): 64%
2m > 3.5 mg/L very high (7-10 points): 23%
4 points
del(17p) / mutated TP53
2m, serum Beta 2 microglobulin; ALC, absolute lymphocyte count; ULN, upper limit of normal; OS, overall survival; del(17p),
deletion of short arm of chromosome 17; del(11q), deletion of long arm of chromosome 11; LDH, lactate dehydrogenase; TTFT,
time to first treatment; TK, serum thymidine kinase; U-CLL, unmutated immunoglobulin heavy chain variable region gene; ECOG,
Eastern Cooperative Oncology Group performance status.
** Formula for calculating risk score is as follows: [I(No. of lymph node sites involved =3) 7.370 + I(del11q) 9.312 + I(del17p)
11.285 + (diameter of largest cervical lymph node in cm) 4.172 + (LDH/100) I([IGHV gene = mutated] 5.000 + (LDH/100)
I(IGHV gene = unmutated) 1.065] + 35.467. The indicator function (I) is equal to 1 if the statement in the parentheses is true and
equal to 0 otherwise.
90 Nili Saar, Pia Raanani and Uri Rozovski

MICRORNA'S
MicroRNAs (miRNAs) are a class of small non-coding RNAs that
function as post-transcriptional regulators of gene expression [188]. The first
report linking microRNAs to cancer was in CLL, when a cluster of 2
microRNAs, miR-15-a/miR16-1 was found to reside in the minimally deleted
region of patients with CLL and 13q14 deletion [189]. This cluster is
downregulated in approximately 70% of patients with CLL and results in
increased levels of BCL2, which provide CLL cells relative protection from
apoptosis [190]. The miR-15-a/miR16-1 cluster is only one of many
microRNA clusters that are dysregulated in CLL [191]. MicroRNA profiles
have been found to correlate with other prognostic markers [192] as well
as with disease progression [193-198], response to treatment [199-201] and
survival outcome [192]. Plasma levels of miR155, for example, predicted
progression of monoclonal B-cell lymphocytosis to overt CLL [201], and
high levels predicted poor response to chemo-immunotherapy [201, 202].
Nonetheless, most expression signatures were not validated in separate cohorts
and different studies yielded inconsistent, sometimes contradictory results,
making it difficult to recommend any specific microRNA as a reliable
biomarker in clinical practice.

CLOSING REMARKS: USING BIOMARKERS ON


THE ROAD TO CURE

The huge variability in clinical presentation, response to treatment and


survival outcome of patients with CLL led to the development of numerous
prognostic indicators, and several attempts to integrate them into prognostic
scales that predict survival outcome or time to first treatment (Table 2). In a
disease once considered incurable, long term follow up of patients treated with
chemo-immunotherapy revealed that a fraction of these patients experience
prolonged remission duration of 10 years or longer, and are practically cured
[203, 204]. The introduction of oral BTK-inhibitors which demonstrate
efficacy even in high risk CLL patients, with a significantly superior safety
profile compared to chemotherapy, but do not lead to cure, added another layer
of complexity. Choosing between these two strategies, the good and the
better, has become a reality in treating treatment-nave CLL patients.
Predictive factors that could stratify patients on the basis of how likely they
Prognostic and Predictive Indicators in Chronic Lymphocytic 91

are to benefit from an intent to cure therapeutic strategy, could help


tailor treatment. Specifically, approximately 50% of patients with M-CLL
experience prolonged remissions when treated with FCR. Patients with M-
CLL and low 2M are even more likely to achieve cure, with approximately
60% experiencing prolonged remissions following standard treatment [203].
Conversely, while the overall response rate in elderly patients treated with
ibrutinib was 86% [205], CR was achieved in only 4%, making CR or cure
unrealistic therapeutic goals with this modality. It is therefore reasonable to
recommend FCR treatment for patients with M-CLL and low 2M levels, and
a novel drug, such as ibrutinib, for patients with U-CLL and high 2M levels
who are not likely to get cured with FCR. Determining IGHV mutation status
is therefore imperative and recommended by us and others [66, 206] in all
newly diagnosed patients with CLL.

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In: Chronic Lymphocytic Leukemia ISBN: 978-1-63485-510-5
Editor: Kimberly Rodriquez 2016 Nova Science Publishers, Inc.

Chapter 4

MOLECULAR CYTOGENETIC
ABNORMALITIES IN CHRONIC
LYMPHOCYTIC LEUKEMIA:
PROGNOSTIC IMPLICATIONS

Prabhjot Kaur, MD
Associate Professsor of Pathology, Department of Pathology,
Dartmouth Hitchcock Medical Center and
Geisel School of Medicine at Dartmouth 1 Medical Center Drive
Lebanon, NH, US

ABSTRACT
CLL is the most common leukemia in the western world. Mostly a
disease of the elderly, this indolent disorder, has a very variable clinical
outcome and can evolve into an aggressive lymphoma. As indolent
disorders are considered incurable, treatment is delayed until the patient
progresses to high stage disease. These therapeutic decisions are based on
criteria devised by Kanti Rai and Jacques Louis Binet almost four
decades ago; this assessment requires only a physical examination and a
complete blood count. A significant change that has occurred in the past
four decades is the increased diagnosis of patients in low stage disease
(during regular check-ups for employment or other causes). In the 1970s,
40% of the patients were diagnosed at stage I. In the 2000s, nearly 80%
of the patients are diagnosed at low stage. Although patients are
diagnosed at an earlier stage, it is difficult to determine the prognosis of
116 Prabhjot Kaur

Rai stage 0/1 patients. A subset of patients with early stage CLL will
rapidly evolve to a more aggressive and fatal disease, and clinical staging,
as it currently exists, fails to predict this progression.
Approximately 80% of individuals with CLL have acquired
chromosomal abnormalities within their malignant clone and can be
categorized into five prognostic groups accordingly: deletion 13q (median
survival, 133 months); deletion 11q (median survival, 79 months);
trisomy 12 (median survival, 114 months); normal cytogenetics (median
survival, 111 months); and deletion 17p (median survival, 32 months)
(Dhner et al., 2000). A complex cytogenetic karyotype can be identified
rarely and is commonly associated with poor prognostic features
including CD38 expression and unmutated IgHV (Haferlach et al., 2007).
IGHV3-21 usage is an independent poor prognostic factor. Novel
mutation in CLL include the NOTCH1 mutations (Puente et al., Nature
2011, Rossi et al., Blood 2012), SF3B1 mutations (Quesada et al., Nature
Gen 2012), BIRC3 mutation (Rossi et al., Blood 2012) among others. B
cell receptors play a unique role in the pathogenesis of the disease and
has prognostic and therapeutic implications. Stereotyped B cell receptors
impact outcome. New hierarchal classification is being suggested to
include the conventional cytogenetic markers and novel mutations.
MicroRNAs (miRNA) are short (~22nt in humans), endogenous non-
coding ssRNA (single stranded) molecules that regulate gene expression
via translational repression or transcript degradation. Recent evidence has
shown that miRNAs are key to gene regulation and have a global effect
on various oncogenic, tumor suppressor and cell survival pathways. Over
the last decade several miRNAs have been shown to be involved in the
pathogenesis of CLL. In this chapter, molecular cytogenetic prognostic
markers, BCRs, novel mutations, role of miRNA in the biology and
prognosis of this disease are discussed.

INTRODUCTION
Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in
the western world, seen mostly in the elderly age-group and has a very
variable clinical outcome. Traditionally considered an indolent, antigen
inexperienced leukemia of slowly accumulating cells that do not die, we now
acknowledge that CLL cells are highly proliferative, antigen experienced cells
that have a high cell turnover and a subset show an aggressive clinical course.
The aggressive behavior seen argues against the notion of a low proliferative
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 117

disease. In vivo studies using nonradioactive means suggest that CLL cells are
more dynamic than is usually appreciated. CLL cells have replication rates,
ranging from about 0.1 to more than 1.0 percent of the clone per day [1]. If the
total clonal burden of a typical patient with CLL is approximately 1012 cells,
these rates point to the daily production of some 109 to 1010 new leukemic
cells! [2]. Another traditional view that B-CLL is a disease primarily resulting
from the accumulation of clonal B lymphocytes that do not die has been
challenged by the evidence provided by telomere length which is an index of
cell replication. In a study by Damle et al. where telomere length was
quantified by a flow-FISH hybridization protocol, it was found that B-CLL
cells have shorter telomeres than normal age-matched B cells suggesting that
leukemic cells have extensive proliferative histories [3]. A significant change
that has occurred in the past four decades is the increased diagnosis of patients
in low stage disease mostly during regular check-ups for employment or other
causes. In the 1970s, 40% of the patients were diagnosed at stage I. In the
2000s, nearly 80% of the patients are diagnosed at low stage [4]. It is
especially difficult to determine the prognosis of Rai stage 0/1 patients. A
subset of patients with early stage CLL will rapidly evolve to a more
aggressive and fatal disease [5, 6]. As indolent disorders are considered
incurable, treatment is usually delayed until the patient progresses to high
stage disease. These therapeutic decisions, till recently, were based on criteria
devised by Kanti Rai and Jacques Louis Binet almost four decades ago; an
assessment that requires only a physical examination and a complete blood
count. Some patients may benefit from early treatment. However therapies are
not without toxicities and earlier clinical trials showed that treatment in early
stage disease did not improve survival [7]. Several prognostic markers based
on genetic, phenotypic, and molecular characteristics have emerged in the past
decade. The clinical utility of these newer prognostic indicators, alone or in
combination with each other and other clinical predictive systems are a subject
of active research. The cytogenetic evaluation has evolved tremendously since
the 1970s from conventional karyotyping on metaphase spreads, FISH analysis
on interphase chromosomes, microarrays, SNP arrays for copy number
measurement, DNA methylation to the new era of next generation sequencing
that encompasses whole genome/exome sequencing and targeted sequencing.
Indeed molecular genetics has traversed an exciting journey and Chronic
Lymphocytic Leukemia exemplifies it.
118 Prabhjot Kaur

CHROMOSOMAL ABERRATIONS IN CLL


Cytogenetic abnormalities are seen in a significant subset of CLL.
See Table 1. In the 1970s they were primarily studied by conventional
karyotyping. Chromosome banding is difcult in CLL as the lymphoma cells
show poor mitotic activity that result in poor quality metaphase spreads [8].
The introduction of B-cell mitogens such as TPA (12-O-tetradecanylphorbol-
13-acetate), lipopolysaccharide, pokeweed, antihuman IgM, and B-cell growth
factor [9, 10, 11], all of which successfully stimulated in vitro growth of B
cells, allowed the identication of recurrent chromosome aberrations and by
the 1990s, aberrations were reported in 4050% of the CLL cases [12, 13].
More recently, improved detection rate of cytogenetic abnormalities have been
reported with the use of CpG-oligonucleotide DSP30 and interleukin 2
stimulation [14]. Immunological techniques in parallel with cell culture in B-
CLL have shown that despite B cell mitogens the majority of analyzable
metaphases obtained represent the non-malignant T-cell population [15]. In the
1990s, Fluorescence in situ hybridization (FISH) technology allowed
increased sensitivity of detection of chromosomal aberrations (now found in
more than 80% of cases) and identification of candidate genes. This technique
can be applied to interphase cells and detect abnormalities with a resolution of
several kilobases (kb) as opposed to conventional cytogenetics where the
abnormalities have to be large (megabase pairs) in order to be detected [8] by
chromosomal banding. The most common aberrations detected are deletion of
band 13q14, trisomy 12, deletion 11q22.3-q23.1, deletion 17p13 and del 6q21
[16]. Approximately 50% of these patients show single abnormalities, 25%
display two abnormalities, and the remaining cases demonstrate complex
chromosome changes [13, 17]. Additional aberrations noted in CLL are
trisomies 8q and 3q. Translocation breakpoints, in particular involving the
immunoglobulin heavy chain locus at 14q32, which are frequently observed in
other types of non-Hodgkins lymphoma, are rare events in CLL.
Chromosome 13q. Deletion 13q14.3 is the most common chromosomal
abnormality detected by banding and FISH techniques in CLL, occurring in
over 50% of patients [8, 18, 19]. It presents as the sole abnormality in up-to
45% of CLL, i.e., in the absence of other cytogenetic abnormalities. Del 13q
predicts a more favorable prognosis. Subsequent studies showed that within
this subgroup some patients did not require treatment for years or decades
while others developed aggressive disease. This was associated with increased
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 119

del13q tumor burden where cases that showed greater than 65-85% of their
cells with the deletion showed worse prognosis. This was likely due to the
overexpression of genes involved in proliferation and underexpression of
genes involved in apoptosis in patients with more del (13q) cells [18-22].
Candidate genes in this region that were initially proposed included the
Retinoblastoma gene (RB1), Leu1, Leu2 and BrCA2 genes. The minimal
deleted region (MDR) includes the DLEU2/Mir15A/Mir16B loci, however
there is a large variation in the size of the deletion among patients. [23, 24]
[25-27]. MicroRNAs (miRs) will be addressed separately in this chapter.
Chromosome 11q and ATM as a candidate gene. 11q deletion is the
second most common aberration and reported in 20% of the patients [28-30].
It contains a minimal deleted region of 2-3 Mb at 11q22.3-11q23.1 with ATM,
the candidate tumor suppressor gene. The mutation has been associated with
defective DNA repair [31]. The Bcl-1 locus at 11q13 and the MLL gene at
11q23.3 are located outside the critical region. Patients with an 11q deletion
have a more rapid disease progression with shorter treatment-free periods and
inferior survival [9, 11, 28, 29, 32-35, 36].

Table 1. Chromosomal aberrations in CLL


120 Prabhjot Kaur

Chromosme 12. Trisomy 12 was the first chromosomal abnormality


discovered in CLL. This abnormality was reported at a frequency of 10-20%
by conventional banding [11, 32, 33, 37-41] and in up to 30% of cases by
FISH [40, 42, 43], making it the third most frequent aberration. Partial trisomy
has also been reported and alludes to duplication of a critical region, 12q13-
q22 [39, 40, 44]. It has also been shown that trisomy results from duplication
of one homolog [13]. Trisomy 12 is seen only in a subset of the clonal CLL
cells in most studies suggesting that this may be a secondary event developing
in an already established neoplastic cell population. Trisomy 12 defines a
subgroup of CLL with more frequent atypical morphology including
prolymphocytes, strong surface immunoglobulin and FMC7 expression, more
advanced stages and possibly worse prognosis [13, 41, 43, 45, 46].
Chromosome 6q. Deletion 6q has been detected in 6-7% of evaluable
cases with bands 6q15 and 6q23 most frequently affected. Some studies report
a higher tumor mass/lymphadenopathy without any differences in prognosis or
survival. Del 6q confers an intermediate prognosis [47, 48].
Chromosome 17p13 and TP53 as a candidate gene. Del17p13 has been
reported in 4 to 16% of the cases [31, 39, 49] and show poor survival due to
advanced disease at diagnosis, short time to first treatment, and high risk of
chemorefractoriness to alkylating agents and purine analogues [50, 51]. Like
chromosome 13, here too the tumor burden has been linked to prognosis with
patients with 20% 17p deletion nuclei showing longer median time to first
treatment (TTFT) and overall survival (OS) [19].

Table 2. National Cancer Network Guidelines for prognostication and


treatment of CLL
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 121

TP53 maps to 17p13.1 and is regarded as the candidate tumor suppressor


gene due to del17p13. By single-stranded conformation polymorphism
analysis, direct sequencing of PCR-amplified fragments, and FISH studies,
TP53 abnormalities were found in 10-15% of the cases [30, 31, 52-54]. These
are localized to the DNA-binding domain that is critical for its tumor-
suppressor activity and have been associated with poor prognosis, treatment
failure, shorter survival time, even in multivariate analysis after adjusting for
clinical parameters like age, Rai stage and hemoglobin level. [49, 50, 54, 55-
57]. It has been seen that TP53 mutations can be seen in the absence of del
17p13 in at least 20% of the cases and are associated with similar short
survival and chemorefractoriness [55]. The incidence of TP53 mutations has
been reported to increase as the disease progresses; the incidence rises to
10%12% at first-line treatment and approximately 40% at refractory CLL [5].
The European Research Initiative on CLL (ERIC) recommends TP53
mutational screening (exons 49) for all patients before the administration of
any therapy [58]. TP53 sequencing is also part of National Comprehensive
Cancer Network (NCCN) guidelines. See Table 2.

B Cell Receptors (BCRs)

The BCR includes sIg (IgM) and the Ig-/Ig- heterodimer (CD79a and
CD79b). The B-cell receptor (BCR) signaling pathway is critical to the
development and maturation of normal B cells [59] (See Figure 1). BCR
activation can result from chronic antigenic drive by microbial or viral
antigens to auto-stimulation of B-cells by self-antigens. The sIg subunits bind
antigen, resulting in receptor aggregation, while the / subunits transduce
signals to the cell interior. The BCR on antigenic stimulation leads to the
activation of ITAMs and SRC family kinases including spleen tyrosine
kinase (SYK) and LYN (Figure 1). These, along with adaptor proteins such
as CD19 and B-cell linker (BLNK), and signaling enzymes such as
phospholipase C-2 (PLC2), phosphatidalyinositol-3-kinase (PI3K)-together
called the signalosome - activate multiple signaling cascades. The downstream
effectors can be modulated toward the pro-apoptotic nuclear factor of activated
T cells (NFAT) arm or the pro-survival NF- kB arm depending on balancing
of the signaling cascades. These permit many distinct outcomes, including
survival, tolerance (anergy) or apoptosis, proliferation, and differentiation into
antibody-producing cells or memory B cells. B-cell maturation involves
somatic recombination and mutation of the IGHV genes that encode the
122 Prabhjot Kaur

antigen binding domains of the BCR [59-63]. CLL has distinct BCR signaling
compared with normal B cells [64-67]. CLL cells demonstrate anergy
(decreased or no response to BCR engagement) and this BCR signaling
capacity depends on the mutational status of the immunoglobulin heavy chain
variable (IG V) genes. CLL cells with mutated IGHV genes usually express
lower levels of surface IgM (sIgM) and present a weak response to BCR
engagement associated with indolent disease. In contrast, cells with unmutated
IGHV genes that predominantly express the ZAP-70 kinase, respond to BCR
stimulation and reflect an aggressive clinical behavior [66-69]. BCRs that can
react to multiple antigens (polyreactive) are associated with more aggressive
disease. CLL subsets that show strong intracellular responses demonstrate a
more aggressive clinical course. The microenvironment within a lymph node
supports strong co-stimulatory signals with stronger BCR signaling in contrast
to peripheral blood. The canonical NF-B pathway was significantly up-
regulated in lymph node samples, and the expression of the inhibitory I-Bs
was significantly lower [69-72]. These studies show that the BCR signaling
plays an important role in the pathogenesis of CLL. Several promising
therapeutic advances have been seen via targeting the molecules in the BCR
signaling pathway. Some examples of drugs that inhibit kinases within the
BCR signaling pathway are ibrutinib (an inhibitor of Brutons tyrosine kinase
(Btk)), fostamatinib (an inhibitor of Syk), idelalisib (an inhibitor of
phosphatidylinositol 3 kinase (PI3K)) and Everolimus (an mTOR inhibitor)
[61, 63, 73-79].
The IgHV mutation status. Damle et al. and Hamblin et al. described, in
landmark studies, the prognostic significance of the immunoglobulin heavy
chain variable region gene (IgHV) mutation status in CLL [80, 81]. Mutational
status is determined by comparing the sequence of the CLL cells to the germ
line sequence. A CLL cell is considered as mutated if the deviation is 2% or
otherwise <98% homologous to the corresponding germ line sequence. The
mutation status of the IgH variable region has been used to subdivide CLL into
two groups: one with a proposed pre-germinal center (unmutated) cell origin
and one with a post-germinal center (mutated) cell origin [80, 81]. The group
of CLL cases with unmutated IgH (U-CLL) genes showed rapid disease
progression and a short treatment-free survival [82]. The group with mutated
IgH genes (M-CLL) followed an indolent clinical course with long survival
probabilities. There have been significant differences in the distribution of
cytogenetic abnormalities based on mutation status. The overall incidence of
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 123

genomic aberrations (80%) are similar in the U-CLL and M-CLL subgroups.
The 13q14 deletion, the most common genetic abnormality in CLL, was found
more often in patients with M-CLL, a subset with a more favorable clinical
outcome [80, 81, 83]. High-risk genomic aberrations such as 17p- and 11q-
occurred almost exclusively in the U-CLL subgroup.
The analysis of DNA sequences to determine the status of
immunoglobulin V-gene mutations is laborious and not performed routinely in
clinical laboratories. There has been a search for surrogate markers that can be
studied by immunohistochemistry, flow cytometric analysis or PCR that can
be applied to daily use. CD38 expression, defined as greater than 30%
expression by neoplastic cells, was shown to be a surrogate marker for the
U-CLL genes [84-87]. CD38 promotes survival and proliferation of B cells on
their way to and after neoplastic transformation [88]. CD38 expression
predicts unfavorable prognosis, shorter time from diagnosis to therapy, and
worse overall survival. However, CD38 expression can change over time,
making it difficult to use it as a prognostic marker [85, 89].

Figure 1. The B-cell receptor (BCR) signaling pathway.


124 Prabhjot Kaur

Gene expression analyses found that the zeta-chain associated protein of


70 kDa (ZAP-70), a member of the Syk family of tyrosine kinases and
involved in T-cell receptor signaling [90], was differentially expressed in CLL
with unmutated IgHV genes as compared to CLL with mutated IgHV genes
and that measurements of this protein can be used as a surrogate marker for
expression of U-CLL genes [84, 86, 91-94]. ZaP-70 positivity was associated
with a shorter progression free survival both within normal karyotype and
within the poor-risk cytogenetic subset. In some studies ZAP70 positivity was
associated with aggressive clinical course and short time to treatment, even in
subset of patients with early-stage, asymptomatic disease. Knowledge of the
IGHV mutation status or CD38 did not improve the ability to predict the time
to treatment. In other words, its positivity was independently associated with
aggressive disease. There are reports that have shown discordance of ZAP70
status with mutation status with the discordance highest in cases that carried
additional cytogenetic abnormalities such as 11q deletion, 17p deletion, and
V3-21 usage [95].
More recent gene expression profiling studies show CRY1 and Mboat1 as
potential genes that may serve as surrogate markers for predicting IGHV
mutational status [96]. These have not been significantly explored in clinic
labs and are primarily research results. There are novel immunoglobulin heavy
variable (IGHV) genes and alleles being identified which change the reference
germline library. This has implications on determining the mutation status
which is determined by comparing the nucleotide sequence of the rearranged
IGHV gene with that of the germline sequence. It may mean re-evaluating
cases that are borderline mutated [97].
Stereotype in CLL. Messemer et al. coined the term stereotyped antigen
receptors for the phenomenon of highly similar B cell receptors (BCRs) being
found in CLL cells of unrelated patients. DNA sequencing revealed extremely
restricted use of specific heavy and light chain variable region genes. Within
the heavy and light chain variable regions the complementarity determining
region 3 (CDR3) was particularly conserved with respect to length, amino acid
sequence and unique amino acids near recombination junctions. They
estimated that with random V(D) J gene segment usage and recombination at
junctions between gene segments, the probability of finding the same
combination of heavy and light chain variable genes in independent leukemic
(or normal) B cells would be 1:109 to 1:1012. This points to the intriguing
possibility that the observed similarity between BCRs from different CLL
cases may be antigen-driven. The exact criteria for BCR stereotyping have
evolved slightly over the years. As applied by Messemer, et al. the criteria
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 125

used to define sets of similar rearranged VH DJH were as follows: (a) use of
the same VH, D, and JH germline genes; (b) use of the same D segment reading
frame and position relative to the VH, plus or minus one codon; and (c) an
amino acid identity within the HCDR3 of >=60% identity. More recently,
stereotyping criteria have become somewhat less stringent with respect to
amino acid identity and the IGHV gene used and more focused on amino acid
similarity (functional conservation) and lack of differences in CDR3 length.
Several studies showed the presence of such sterotyped Vh CDR3 sequences
among CLL BCRs and found that at least 30% of CLL cases could be
categorized into one of several subsets based largely on stereotyped amino
acid motifs. A growing body of evidence suggest that stereotyping may be
clinically relevant. For example, subsets of CLL cases with stereotyped BCRs
displayed distinctive features with regard to demographics, immunophenotype,
and outcome [98-101, 102].
IGHV3-21. Tobin et al., reported preferential use of the VH3-21 gene in
mutated CLL and showed that regardless of VH gene mutation status, the
VH3-21 cases showed overall poor survival. A large fraction of VH3-21 cases
also demonstrated shorter lengths of the third complementarity determining
region (CDR3) and noticeable similarities in both the nucleotide sequence and
thus amino acid composition. Further analysis of the light chain V gene use,
revealed a considerable restricted use of the Ig light chain gene V2-14 in the
majority of the VH3-21 cases. This suggest that a common antigen epitope is
recognized by the highly homologous VH3-21/V2-14 Ig molecules and
therefore antigen stimulation may be a promoting factor in the evolution of
certain CLL clones [103, 104, 105-107]. This finding is taken as evidence that
antigens and/or superantigens may be involved in lymphoma development by
stimulating proliferation and expansion of B cells expressing distinctive BCR.
This contradicted another time-held notion that CLL is a disease of antigen
inexperienced B cells [2].

Novel Mutations in CLL

Whole genome/exome sequencing has uncovered novel somatic


mutations in CLL that also contribute to prognostic information and cellular
transformation. The genes containing novel variants include NOTCH1 (10-
17%), SF3B1 (10-14 %), TP53 (7-13%), ATM (8-15%), and MYD88 (3-8 %)
[31, 108-114]. All except MYD88 are associated with un-mutated IGHV
mutation status. The low frequency genetic alterations that are seen at lower
126 Prabhjot Kaur

than 5% frequency (with probable function included in parenthesis): XPO1


(Nuclear export), CHD2 (Chromatin modification), POT1 (Telomere
maintenance), HIST1H1E (Histone protein), NRAS (Cell growth), BCOR
(Apoptosis regulation), ZMYM3 (Chromatin modification), SAMHD1 (Innate
immune response), KRAS (Cell growth), MED12 (Gene transcription),
ITPKB(B-cell signaling), DDX3X (RNA helicase) [31, 109, 110, 114].
NOTCH1 is one of the frequent CLL driver mutations identified in
sequencing studies [31, 109, 110, 115]. The NOTCH1 gene, elegantly
summarized by Rossi et al. [116] encodes a heterodimeric transmembrane
protein that functions as a ligand-activated transcription factor [117, 118] and
plays an important role during embryogenesis and in self-renewing tissues of
the adult organism, including maintenance of stem cells, cell fate specification,
proliferation, and apoptosis. In mature B-lymphocytes, NOTCH1 signaling
promotes terminal differentiation to antibody-secreting cells [119]. One of the
mechanisms of the NOTCH1 signal suppression is operated through the PEST
[proline (P), glutamic acid (E), serine (S), and threonine (T) rich] domain that
directs the activated NOTCH1 towards proteosomal degradation [117]. One
recurrent mutation, a 2-bp frameshift deletion (c.7544_7545delCT) accounts
for approximately 80% of all NOTCH1 mutations [115]. These mutations
generate a premature stop codon, resulting in a NOTCH1 protein lacking the
C-terminal domain, which contains a PEST sequence. This in turn results in
accumulation of an active NOTCH1 isoform, which is associated with a
distinct transcriptional signature [109]. These NOTCH1 mutations were
primarily found in patients with the more clinically aggressive U-CLL gene
subtype of CLL (20.4%) in Richter syndrome (31.0%), and in chemorefractory
CLL (20.8%). Among CLL genetic subgroups, they cluster with cases
harboring trisomy 12 and tend to be mutually exclusive with TP53 disruption.
These results suggest that although NOTCH1 mutations are not
pathognomonic or causative of CLL, they are associated with poor prognosis
and could define a distinct clinical subtype for therapeutic intervention.
NOTCH1 is a potential therapeutic target [117].
SF3B1 is a central component of the U2 spliceosome, which promotes
excision of introns from pre-mRNA to form mature mRNA [120]. It has been
reported that CLL cells with SF3B1 mutations show defective splicing
activity, thus resulting in deregulated normal and alternative mRNA splicing
[121]. Whole genome/exome sequencing technologies showed SF3B1
mutations in 10-17% of the cases [31, 110, 111]. SF3B1 mutations are
associated with faster disease progression and poor overall survival, and seen
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 127

in CLL without IGHV mutation [110]. These mutations were also found in
higher rates in patients that have chemo-refractory CLL [121].
MYD88 gene mutation, a critical adaptor molecule of the Toll-like
receptor (TLR) complex, is seen in 3-10% of CLL cases [31, 109]. The
recurrent MYD88 mutation in CLL (L265P) causes constitutive MYD88-
IRAK signaling, resulting in constitutive NF-B activity. MYD88 L265P
mutations have been found predominantly in M-CLL. This aberration is
potentially amenable to therapeutic targeting through direct inhibition of the
MYD88-IRAK complex, through proteasomal inhibition [122] or even
through the inhibition of Brutons tyrosine kinase (BTK) [123].
BIRC3 gene is a negative regulator of alternative NF-kB signaling
pathway. Its mutations or deletions, noted in less than 5-8% of cases, lead to
the activation of alternative NF-kB pathway [111]. ATM and the BIRC3 genes
are located on 11q. Initial studies speculated that BIRC3 mutations may
explain the poor prognosis of 11q deletion, however ATM mutation rather
than BIRC3 deletion and/or mutation predicts reduced survival in 11q-deleted
chronic lymphocytic leukemia [124]. Targeted sequencing of the BIRC3
coding sequence in CLL showed that BIRC3 inactivation is particularly
common in fludarabine-refractory patients (24%) [111]. BIRC3 disruptions
have been associated with unmutated IGHV gene configuration and 11q
deletion with an inferior progression-free survival [125].

Clonal Evolution and the Driver/Passenger Mutation Model


in CLL

CLL, like most tumors, undergo clonal evolution, implying that


most neoplasms arise from a single cell of origin, and tumor progression
results from acquired genetic variability within the original clone allowing
sequential selection of more aggressive subtypes [126]. In CLL this was
supported by FISH and microarray studies [17, 127] and subsequently in next
generation sequencing studies. The initial population of cells may harbor
driver mutations that will contribute to tumor progression and neutral
passenger mutations. Next-generation sequencing studies display the
genomic complexity of CLL with some genetic mutations seen in higher
frequencies than others-the so called mountains vs hills, both being
pathologically significant [114, 128]. The computational algorithms detect
cancer specific alterations with a high probability of being cancer drivers on
the basis of whether they are present at a significantly higher-than-expected
128 Prabhjot Kaur

rate given the known background mutation rate of the cancer. Studies have
followed patients over time and seen the differences in aberrations depending
on the time of evaluation, that is, whether the cytogenetic study was performed
at diagnosis, months after diagnosis, prior to treatment or following treatment.
The consensus has been that new aberrations are acquired over time and that
the acquisition of new genomic abnormalities usually portend a more
aggressive disease with decreased overall survival. Different cytogenetic
abnormalities show differing impact on clinical outcome. For example del
(17p) and/or del (11q) are associated with worse prognosis. The appearance of
new abnormalities is not restricted to cases that have undergone therapy [129],
though treatment can result in disease progression due to expansion of
previously present subclones [130-132]. SF3B1, not usually seen in
monoclonal B cell lymphocytosis (MBL) [133], a clonal condition that is
thought to precede CLL, may be an example of a driver mutation that evolves
from a minor sub-clone to a dominant sub-clone during disease progression or
relapse [74, 75] and therefore may have a role in clonal evolution in CLL [2,
134]. The incidence of TP53 mutations has been reported to increase as
the disease progresses; the incidence rises to 10%12% at first-line treatment
and approximately 40% at refractory CLL [5]. NOTCH1 and P53 are
mutually exclusive, whereas are seen in combination at the time of Richters
transformation of CLL. Emergence of NOTCH1, SF3B1, and BIRC3
abnormalities in addition to TP53 and 11q22-q23 lesions have been seen
changing prognostic categorization from lower to higher risk groups [135].
Clonal evolution can traverse different routes based on tumor heterogeneity or
acquisitions of new mutation during the wait and watch time period or some
driver mutations are positively selected post treatment and expand [129, 131,
136]. See Figure 2.

Figure 2. Model of Clonal Expansion.


Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 129

Hierarchal Classification

CLL shows multiple cytogenetic abnormalities which have prognostic


implication. The Dohner hierarchical classification based on FISH studies,
initially defined in 2000, holds true today [19, 135, 137, 138]. The
chromosomal abnormalities are categorized into five prognostic groups:
deletion 13q (median survival, 133 months); normal cytogenetics (median
survival, 111 months); deletion 11q (median survival, 79 months); trisomy 12
(median survival, 114 months) and deletion 17p (median survival, 32 months)
(Dhner et al., 2000). Karyotypic complexity and a high proportion of
abnormal metaphases are adverse prognostic indicators [12, 139-141]. Most of
the risk models are based on evaluation of risk factors detected at a specific
time point. By integrating mutational and cytogenetic analysis and using
both a training-validation and a time-dependent design, Rossi et al. delineated
four CLL hierarchical subgroups [135], that maintain prognostic relevance
in a time-dependent fashion: High-risk, harboring TP53 and/or BIRC3
abnormalities (10-year survival: 29%); Intermediate-risk, harboring
NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 (10-year survival:
37%); Low-risk, harboring +12 or normal genetics (10-year survival: 57%);
and Very low-risk, harboring del13q14 only (10-year survival 69.3%).
Baliakis et al. [132] proposed that a unifying model for all groups of patients
in all situations may be most desired but difficult to attain. A more realistic
model may focus on patient subgroups at different relevant decision points,
that is at diagnosis and at initiation of first-line or subsequent treatments.

MicroRNAs

MicroRNAs (miRs) are short (~22 nt in humans), endogenous non-coding


ssRNA molecules that regulate gene expression by degrading mRNAs or
inhibiting translation to protein. miRs are key to gene regulation and have a
global effect on various oncogenic, tumor suppressor and cell survival
pathways. Earlier work delineated miRs that were differentially expressed in
CLL cells vs normal cells, the so called signature miRs. These included
miR-16-1, miR-26a, miR-206, and miR-223, miR-155, miR-21, miR-150,
miR-92 and miR-222, miR-181, miR-30d and let-7a [142-144]. Some miRs,
for example miR-15a, miR-195, miR-221, miR-23b, miR-155, miR-223,
miR29a-2, miR-24-1, miR-29b-2, miR-146, miR-16-1, miR-16-2, and miR-
29c showed potential as prognostic markers as they were differentially
130 Prabhjot Kaur

expressed between U-CLL (unmutated IgVH)/ZAP70+ and M-CLL (mutated


IgVH )/ZAP70-CLL patients, or correlated with clinically aggressive disease
[142-145]. The most promising miR connection with CLL is the seminal
finding that the del 13q14 region contains the deleted in leukemia (DLEU) 2
gene and the miR-15a/miR-16-1 cluster [146]. Cimmino et al. (2005) proposed
that miR-15a and miR-16-1 function as tumor suppressor genes by modulating
BCL2 (B-cell CLL/lymphoma 2), an anti-apoptotic protein that is highly
expressed in CLL. Specifically, by analyzing homology between these two
miRs and the BCL2 mRNA sequence, Cimmino et al. found that the first nine
nucleotides from the 5 ends of both miRs are complementary to the BCL2
cDNA. Whereas in normal CD5+ tonsils the levels of both miRNAs were high
and the BCL2 protein was expressed in low levels, the opposite was true in
leukemic cells. Hence miR-15a and miR-16-1 produce their anti-tumorgenic
effect by targeting the BCL2 gene. TP53 maps to 17p13.1 and is regarded as
the candidate tumor suppressor gene due to del17p13. In 2007 several groups
demonstrated that members of the miR-34 family are direct p53 targets. miR-
34 genes are up-regulated by p53, and their over-expression in turn causes
senescence, apoptosis, or cell cycle arrest by regulating proteins such as
BCL2, Cyclin D1, Cyclin E2 depending on the cell type and further to this
studies suggested that p53 mutations/deletions lead to low miR-34a expression
and hence are associated with impaired DNA damage response, apoptosis, and
fludarabine refractory CLL disease [147, 148]. It was suggested in some
studies that miR-34a has the potential to be a surrogate marker for any
deregulation in the p53 pathway. miR-29 has been described as a tumor-
suppressing miRNA which targets several oncogenes including TCL1 [149].
Pekarsky et al. (2006) showed that TCL1 protein expression in CLL was
inversely correlated with miR-29 and miR-181 expression and that these
microRNAs may be candidates for therapeutic agents in CLLs overexpressing
Tcl1l. These have not been validated further for clinical use and remain in
research paradigm.

CONCLUSION
CLL represents a biologically dynamic and heterogeneous disease.
Advances in the basic sciences have led to a better understanding of this
disease process, better markers for prognosis and novel targets for
development of therapies. However development of prognostic models that
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 131

can be universally accepted and applied remain a challenge and are a subject
of ongoing research.

ACKNOWLEDGMENTS
I am grateful to Dr. G J Tsongalis (Professor, Dept of Molecular
Pathology, DHMC) for reviewing the manuscript. Dr. J Lefferts (Dept of
Pathology), DHMC, kindly assisted in editing the chapter.

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INDEX
antagonism, 20
# antibody, 4, 24, 28, 83, 91, 121, 126, 144
anti-cancer, 74
17p13.1 deletion, 19
anticancer drug, 66
3-hydroxy-3-methylglutaryl-coenzyme A
anticonvulsant, 41, 59
(HMG-CoA) reductase, 39
antigen, 31, 36, 44, 52, 83, 100, 101, 104,
105, 116, 121, 124, 125, 141, 142, 143
A antitumor, 37, 41, 48, 53
apoptosis, 3, 16, 18, 25, 28, 29, 30, 31, 32,
ABT-199, 33, 53, 54 33, 34, 35, 38, 39, 40, 41, 50, 51, 55, 58,
ABT-263, 34, 54 59, 60, 62, 64, 66, 80, 90, 102, 111, 119,
Acalbrutinib, 21 121, 126, 130, 147
acetylation, 59 apoptosis pathways, 41
acetylcholinesterase, 67 arrest, 55, 57, 58, 130
acid, 16, 41, 44, 59, 60, 68, 125 arthritis, 23, 24, 48, 49
ACP-196, 21, 22, 47, 48 assessment, 48, 64, 96, 115, 117
acquisitions, 128 asymptomatic, 17, 124
acute lymphoblastic leukemia, 31 ataxia, 34, 81, 86, 108
acute myeloid leukemia, 59 Atorvastatnin, 40
adhesion, 9, 19, 31, 46, 48, 50, 94 ATP, 24, 31, 68, 72
ADP, 103 autoimmune disease, 24, 53
adverse event, 20, 22, 27, 29, 30, 32, 34 autoimmune hemolytic anemia, 40, 59
age, 3, 4, 17, 19, 81, 88, 89, 92, 102, 103,
116, 117, 121
aggregation, 60, 70, 121 B
aggressive behavior, 116
B Cell Receptors, 121
allele, 85, 86, 108, 110
basophils, 3
alters, 58
B-Cell Receptor (BCR) Inhibitors, 18
amino acids, 124, 125
BCL2 family, 33, 34, 35, 38, 40
anatomy, 94
BCL2 Inhibitors, 33
anemia, 21, 30, 34, 80
150 Index

Belarus, 66 cell cycle, 35, 38, 55, 57, 58, 83, 85, 130,
benefits, 34 133
benign, 80 cell death, 38, 57
biological activity, 34 cell fate, 126
biological sciences, 65 cell lines, 25, 38, 43, 66, 69, 102
biomarkers, 111 cell signaling, 126
biomaterials, 75 cell surface, 18, 83, 84, 94
biomolecules, 75 CEP-18770, 36
blood, 1, 8, 17, 28, 33, 68, 70, 74, 80, 114, ceramide, 25
132, 140 CGI-1746, 22, 23
blood cultures, 132 CGI-560, 22, 23
bonds, 8, 42 chemical, 16, 28, 42
bone, 2, 10, 21, 27, 29, 40, 47, 80 chemokine receptor, 1, 3, 5, 6, 8, 106
bone marrow, 2, 10, 27, 29, 40, 80 chemokines, 2, 3, 10, 31
bortezomib, 35, 55, 144 Chemokines, 1, 2, 3, 8, 10, 11, 31
brain, 71 chemotaxis, 29, 30, 52
branching, 42 chemotherapeutic agent, 58
breast cancer, 25, 56, 58, 100 chemotherapy, 33, 36, 43, 51, 55, 58, 59,
Bulgaria, 1, 3, 11 78, 84, 90, 108, 109
cholesterol, 39, 58
cholesterol lowering agents, 39
C Chromosomal Aberrations, 118
chromosomal abnormalities, 95, 97, 116,
cancer, 3, 10, 11, 34, 35, 37, 39, 42, 46, 50,
129, 132
51, 53, 56, 57, 63, 66, 70, 72, 73, 90,
chromosome, 19, 22, 81, 85, 86, 89, 95,
101, 107, 108, 111, 112, 113, 127, 134,
112, 118, 120, 132, 133, 134, 135, 136,
137, 144
147
cancer cells, 34, 42, 53, 73
chromosome bands, 135
cancer progression, 63
classes, 27, 33
cancer therapy, 57
classification, 93, 108, 116, 129, 132, 133,
candidates, 83, 86, 112, 130, 134, 146
138
carbon, 42
cleavage, 41, 84, 86
carcinoma, 100
clinical oncology, 96, 99, 100, 103, 108
carfilzomib, 21, 35, 36, 47, 55, 56
clinical presentation, 90
cascades, 39, 121
clinical trials, 18, 20, 22, 28, 29, 30, 34, 37,
categorization, 128
38, 83, 86, 89, 117
CC-292, 20, 21, 47, 48
clone, 18, 116, 117, 127, 133
CCR, 7, 10
clusters, 11, 90
CCR3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
CNX-774, 22, 24, 25
CCR3+ B-cells, 2, 6, 7
coding, 90, 111, 116, 127, 129, 143
CD193, 3, 4, 7, 8
codon, 86, 125, 126
cDNA, 64, 130
coenzyme, 39, 58
cell biology, 55, 105
colitis, 28
cell culture, 118
Index 151

collagen, 24, 48, 71 cytotoxicity, 39, 40, 41, 42, 47, 59, 66, 68,
colon, 58 81
colon cancer, 58
colorectal cancer, 145
combination therapy, 28, 47, 50
D
community, 45
Danazol, 16, 40, 59
comorbidity, 54
Dasatynib, 32
complementarity, 124, 125
data analysis, 43
complete blood count, 115, 117
DCA, 38
complexity, 90, 127, 129
defects, 44
composites, 75
degradation, 35, 43, 116, 126
composition, 58, 73, 125
dehydration, 32
compounds, 16, 18
Delta, 50, 144
conditioning, 98
Dendrimers, 42, 60, 61, 63, 64, 65, 66, 67,
configuration, 127
68, 69, 70, 71, 72, 73, 74, 75
consensus, 128
dendritic cell, 66
conservation, 125
deregulation, 112, 130, 146
control group, 2, 3, 5
detection, 8, 96, 97, 105, 118, 132, 137
cooperation, 31
deviation, 122
correlation, 3, 5, 6, 7, 8, 9, 32, 98, 135, 145,
diabetes, 16, 56
146
diabetic patients, 37
correlations, 1, 2, 5, 6, 9, 98, 135, 142
diarrhea, 22, 28, 31
corticosteroids, 40
discordance, 124, 141
cosmetic, 74
disease activity, 84, 103
covalent bond, 24
disease progression, 31, 38, 55, 80, 84, 90,
covering, 61
94, 95, 96, 101, 104, 105, 109, 110, 119,
creatinine, 102
122, 126, 128, 134, 137, 140
critical analysis, 93
diseases, 16, 23, 24, 25, 33, 37, 139, 144
Croatia, 75
disorder, 59, 115
crystallinity, 73
distribution, 5, 83, 122
cure, 61, 78, 79, 90
diversification, 143
cycles, 29, 34, 35, 36
DNA, 34, 40, 60, 66, 68, 77, 82, 83, 85,
cyclophosphamide, 44, 78, 81, 91, 92, 95,
117, 119, 121, 123, 124, 130, 137
102, 113
DNA damage, 66, 130, 137
cysteine, 19, 21
DNA repair, 119
cytochrome, 33, 41
DNA sequencing, 124
cytogenetics, 77, 97, 98, 116, 118, 129, 132,
DOI, 12, 63, 68, 69, 73, 74
146
dosing, 23, 29, 56
cytokines, 31
down-regulation, 38, 93, 111, 112, 147
cytometry, 4, 5, 11, 84, 105, 106
drug carriers, 74
cytoplasm, 17
drug delivery, 42, 75
cytostatic drugs, 17
drug design, 42
drug resistance, 39, 42, 49, 109, 137
152 Index

drugs, 15, 16, 19, 36, 37, 43, 48, 61, 62, 64, first generation, 34
66, 78, 81, 86, 122 flavonoids, 35, 55
DSC, 64 flavopiridol, 36, 81
Duvelisib, 29 fluorescence, 80, 93, 104, 133, 135, 136,
dysplasia, 40 146
dyspnea, 30, 32 Food and Drug Administration (FDA), 17,
26, 35, 36, 43
formation, 40, 60, 70
E Fostamatinib, 31, 52, 122
fragments, 121
elaboration, 23
frameshift mutation, 86
embryogenesis, 126
France, 9, 133
employment, 115, 117
FTIR, 74
endometriosis, 16, 40
functional changes, 139
England, 141, 143
Entospletinib, 31, 53
environment, 10, 28 G
enzyme, 23, 24, 27, 39, 74, 83, 121
eosinophils, 3, 10 GDC-0199, 33, 54
epigenetic silencing, 112 GDC-0834, 22, 23, 48
epilepsy, 16 GDC-094, 30, 51
ERA, 66 gene expression, 42, 49, 62, 90, 109, 110,
erythrocytes, 70, 74 116, 124, 129, 137, 139, 141
euphoria, 34 gene regulation, 116, 129
Europe, 61, 80 gene silencing, 66
evidence, 18, 37, 60, 106, 116, 117, 125, gene therapy, 74, 75
134, 135, 142 genes, 17, 41, 43, 49, 79, 80, 85, 91, 93, 99,
evolution, 94, 125, 127, 133, 134, 137, 145, 107, 111, 113, 118, 119, 121, 122, 123,
146 124, 125, 127, 130, 133, 134, 140, 141,
excision, 126 142, 143, 147
exons, 86, 121 genetic alteration, 125
exposure, 34 genetic defect, 144
extracellular matrix, 84 genetic mutations, 127
genetics, 61, 62, 97, 98, 107, 117, 129, 132,
146
F genome, 107, 108, 111, 117, 125, 126, 143
genomic instability, 85
family history, 17
genomics, 94, 144
family members, 33, 34, 35
germ line, 122
FAS, 41
gland, 40
FDA approval, 17, 36
glioblastoma, 57
fever, 17, 27
glucocorticoid receptor, 17
fibrillation, 69, 73
glucocorticoids, 17, 44
fibroblasts, 3, 38
glucose, 37, 56
fibrosis, 59
Index 153

glutamic acid, 126


glycolysis, 38
I
grouping, 80
Ibrutinib, 16, 18, 19, 20, 21, 22, 28, 29, 30,
growth, 18, 41, 56, 57, 84, 108, 118, 126
34, 40, 45, 46, 47, 50, 51, 54, 82, 83, 86,
growth arrest, 41
91, 96, 97, 98, 100, 102, 109, 113, 114,
growth factor, 57, 84, 118
122, 139
growth signal, 56
Idelalisib, 16, 27, 28, 30, 49, 50, 122
GS-1101, 27
identification, 51, 94, 118, 134
GS-9973, 31, 53
idiopathic, 59
guardian, 108
idiopathic thrombocytopenic purpura, 59
guidelines, 50, 80, 92, 121, 131
IgHV mutation, 122
GX15-070, 34, 54
IGHV mutation status, 78, 82, 91, 124, 125
immune response, 50, 126
H immunofluorescence, 4
immunoglobulin, 18, 44, 54, 77, 79, 82, 89,
hairy cell leukemia, 135 91, 99, 104, 105, 106, 118, 120, 122,
harmonization, 105 123, 124, 131, 140, 141, 142, 143
HDAC, 41, 59 immunohistochemistry, 84, 123
headache, 22 immunomodulatory, 36, 82, 86
hematologic neoplasms, 135 immunomodulatory agent, 86
hematology, 11, 40, 61, 95, 106, 108, 132, immunotherapy, 70, 77, 78, 79, 81, 85, 90
139, 144 improvements, 34
hemoglobin, 34, 80, 121 in situ hybridization, 80, 93, 118, 133, 135,
hepatocellular carcinoma, 58 136, 146
hepatomegaly, 17 in vitro, 8, 21, 28, 29, 30, 32, 35, 38, 40, 41,
heterogeneity, 96, 128, 144 43, 48, 55, 58, 59, 61, 63, 68, 70, 102,
histone, 34, 41, 60 118
histone deacetylase, 41, 60 in vivo, 21, 35, 41, 46, 47, 48, 63, 66, 70,
HIV, 57, 66, 67, 68, 70, 72, 75 112, 113, 147
HIV-1, 66, 72 incidence, 17, 44, 56, 81, 92, 121, 122, 128
HM-71224, 22, 24 indirect effect, 56
hormone, 17, 40 individuals, 113, 116
human, 10, 21, 25, 31, 48, 57, 58, 70, 72, indolent, 2, 27, 29, 35, 55, 78, 115, 116,
85, 101, 102, 108, 137, 145 117, 122
hybridization, 117 induction, 32, 33, 34, 35, 40, 44, 46, 55, 59
hydrolysis, 48 infection, 66
hydrophobicity, 68 inflammation, 24, 52
hypercholesterolemia, 16 inflammatory disease, 50
hypersensitivity, 49 informed consent, 3
infrared spectroscopy, 74
inguinal, 80
154 Index

inhibition, 16, 18, 19, 20, 21, 23, 28, 30, 32, ligand, 9, 24, 86, 110, 126, 141
33, 34, 35, 37, 44, 48, 50, 54, 57, 113, linear polymers, 60
127 liposomes, 42, 72, 74
inhibitor, 16, 17, 18, 20, 21, 22, 23, 24, 25, liver, 80
27, 28, 29, 30, 31, 32, 34, 45, 46, 47, 48, loci, 119, 135
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 82, locus, 118, 119, 134
86, 97, 122, 139 low risk, 3, 5, 6
initiation, 110, 129 LTD, 22, 30
inositol, 27 Luo, 100
institutions, 61 lymph, 2, 17, 19, 28, 29, 32, 36, 41, 56, 80,
insulin, 37, 69, 70, 72 81, 85, 89, 94, 106, 120, 122, 139
integration, 56 lymph node, 2, 19, 28, 29, 32, 36, 41, 56,
integrin, 31, 84, 106 80, 85, 89, 106, 122, 139
interface, 73 lymphadenopathy, 17, 80, 81, 94, 120
interphase, 117, 118, 135, 146 lymphocytes, 2, 3, 4, 5, 6, 8, 9, 38, 39, 55,
intervention, 126 57, 59, 78, 80, 91, 117, 126, 144
introns, 126 lymphocytosis, 17, 19, 46, 80, 90, 113, 128,
IPI-145, 29, 50 132, 145
irradiation, 92 lymphoid, 2, 17, 28, 46, 53, 54, 109, 133,
isozyme, 83, 101 135, 137, 138
lymphoid organs, 2
lymphoid tissue, 133
K lymphoma, 1, 3, 9, 10, 11, 17, 19, 20, 25,
26, 27, 29, 31, 34, 35, 36, 44, 45, 51, 52,
karyotype, 82, 96, 98, 116, 124, 137
53, 54, 55, 56, 84, 86, 94, 101, 108, 109,
karyotyping, 82, 98, 117, 118
115, 118, 125, 130, 134, 136, 137, 139,
kinase activity, 32
144, 145
kinetics, 23, 131
LYN Inhibition, 32
LYN kinase activity, 32
L lysine, 67
lysis, 33
lactate dehydrogenase, 41, 89
laser radiation, 70, 71
Leahy, 135 M
Lebanon, 115
mAb, 46
lesions, 128
macromolecules, 42, 60
leucine, 86
macrophages, 52
leukemia, 2, 9, 15, 17, 32, 34, 44, 46, 51,
magnesium, 68
61, 62, 63, 78, 86, 92, 93, 95, 96, 97,
magnetic resonance, 75
109, 115, 116, 130, 131, 133, 134, 135,
magnitude, 33
144, 145, 146
MAI, 104
leukocytes, 5
majority, 18, 118, 125
leukocytosis, 17
malignancy, 139
LFM-A13, 22, 25, 49
Index 155

malignant cells, 41 multiple myeloma, 21, 47, 56


mantle, 20, 31, 35, 45, 55, 84, 134, 139 multivariate analysis, 101, 121, 146
marrow, 80 mutation, 20, 47, 60, 78, 82, 85, 86, 91, 95,
mass, 120 99, 104, 105, 107, 109, 111, 116, 119,
mast cells, 3, 10 121, 122, 124, 125, 126, 127, 128, 136,
matrix, 71 137, 140, 141, 143, 144
maturation process, 18 mutation rate, 128
measurement, 117, 124, 131 myelodysplastic syndromes, 40
median, 3, 5, 17, 19, 21, 27, 29, 32, 33, 34, myelofibrosis, 34
36, 41, 81, 116, 120, 129
medical, 2, 62, 75
medication, 16, 38
N
medicine, 66, 93, 103, 111, 114, 131, 132,
nanomedicine, 71
134, 139, 141, 143, 144, 145
nanoparticle, 16, 42, 63, 71, 72, 73
Mediterranean, 139, 144
nanosystems, 75
membranes, 42, 65
National Academy of Sciences, 93, 101, 111
memory, 121
natural killer cell, 84
memory B cells, 121
Navitoclax, 33, 34, 54
meta-analysis, 56, 114
necrosis, 35
metabolism, 37
neoplasm, 2
metaphase, 98, 117, 118, 135, 146
nephritis, 26
metastasis, 11
nerve, 69
metformin, 16, 37, 56, 57
Netherlands, 24
methylation, 106, 117
neuroblastoma, 69
methylprednisolone, 46
neurodegenerative disorders, 66
Mevinolin, 58
neuronal cells, 67
MHC, 83
neutral, 109, 127
mice, 21, 24, 31, 53
neutropenia, 20, 22, 29, 30, 31, 32, 36
microRNA, 90, 112, 146, 147
New England, 93, 95, 96, 97, 104, 106, 107,
migration, 1, 2, 8, 10, 27, 31, 48, 52, 71,
109, 110, 114, 131, 132, 134, 139, 141,
104, 106
145
MIP, 24
New Zealand, 103
mitogen, 82, 97, 98, 118, 132, 135
next generation, 85, 117, 127
MLN9708, 36
NF-kB Targeting Agents, 35
models, 22, 26, 29, 48, 50, 52, 129, 130
NF-B signaling, 35, 43, 127
molecular biology, 15, 61, 62
NH2, 70
molecules, 9, 17, 31, 35, 83, 94, 101, 116,
NMR, 72
122, 125, 129
nodal involvement, 94, 134
monoclonal antibody, 4
non-Hodgkins lymphoma, 83, 118
monomers, 42
nucleic acid, 42, 65, 66, 68, 75
morphology, 66, 75, 120, 135, 136
nucleoside analogs, 40, 59
mortality, 37, 56
nucleotide sequence, 77, 82, 124, 125
mRNA, 24, 41, 84, 105, 107, 126, 129, 130
nucleotides, 130
156 Index

nucleus, 36, 86, 120 phenotype, 40, 51


phosphatidylserine, 39
phosphorus, 65, 67, 68, 69, 70, 71, 72, 73
O phosphorylation, 21, 27, 30, 31, 32, 35, 38,
43, 48, 51, 83
Obatoclax, 34, 54
photosensitizers, 74
oligomerization, 33
physiology, 103
oncogenes, 130
PI3 Kinase Inhibition, 27
ONO-4059, 22, 48
PI3K, 16, 27, 28, 29, 30, 43, 50, 51, 121
opportunities, 15
PI3K pathway, 27
optimization, 37
PI3K/AKT, 16, 30, 50
organelles, 42
Pictilisib, 30
organism, 126
pilot study, 38
organs, 33
placebo, 28
orpozomib, 36
platelet count, 34
platelets, 6, 53, 80
P PM, 97, 113, 132, 138
pneumonia, 21, 27, 32
p53, 56, 81, 85, 95, 108, 109, 130, 136, 137, point mutation, 86
147 Poland, 15, 61, 62, 64, 65
pallor, 17 polarization, 73
Pan-PI3K/mTOR Inhibition, 30 polymers, 42, 60, 73, 74
parallel, 118 polymorphisms, 86, 121
patents, 61, 71, 72 population, 4, 103, 118, 120, 127, 133
pathogenesis, 15, 59, 91, 112, 116, 122, positive correlation, 2, 5, 6
131, 146 prednisone, 17, 44
pathology, 97, 101, 103, 132, 144 pregnancy, 41
pathophysiological, 10 prevention, 57
pathophysiology, 81 primary cells, 29
pathway, 16, 17, 19, 27, 30, 31, 33, 35, 36, principles, 144
37, 39, 41, 43, 50, 52, 60, 81, 83, 86, probability, 124, 127
104, 111, 116, 122, 127, 129, 130, 139 prognosis, 15, 38, 39, 77, 81, 83, 84, 85, 86,
PCI-32765, 18, 45 94, 98, 100, 101, 103, 106, 107, 109,
PCR, 84, 121, 123 110, 112, 115, 116, 117, 118, 119, 120,
PCT, 65, 67 121, 123, 126, 127, 128, 130, 134, 136,
pepsin, 70 141, 142, 146
peptides, 66, 67, 68, 70, 72, 74 programming, 57
peripheral blood, 4, 19, 21, 34, 40, 41, 42, project, 61, 64
122 proliferation, 2, 3, 17, 18, 21, 23, 27, 30, 33,
peripheral blood mononuclear cell, 34, 41, 37, 43, 52, 53, 56, 57, 83, 84, 93, 100,
42 101, 102, 104, 109, 119, 121, 123, 125,
pH, 66 126, 138, 139
pharmaceutical, 16, 17, 64 proline, 86, 126
pharmacokinetics, 21, 70
Index 157

propylene, 42, 61, 63, 65, 67, 68, 69, 70, 71, residue, 21, 24, 42
73, 74 resistance, 33, 41, 42, 44, 47, 51, 55, 58, 59,
prostate cancer, 58 63, 78, 84, 85, 109, 113
proteasome, 35, 36, 47, 54, 55, 56 respiration, 56
proteasome inhibitors, 35, 36, 54 response, 18, 19, 22, 26, 28, 29, 32, 34, 36,
protection, 39, 90 41, 46, 60, 63, 78, 79, 81, 82, 83, 86, 90,
protein kinases, 25 96, 102, 108, 122, 130, 132, 136, 137,
protein synthesis, 102 138, 145
proteins, 27, 33, 34, 35, 38, 40, 41, 42, 65, retinoblastoma, 133
66, 68, 73, 74, 121, 130 RG7601, 33
proteolysis, 40 rheumatoid arthritis, 26, 31
proteolytic enzyme, 66 risk, 2, 3, 4, 5, 6, 7, 8, 9, 16, 17, 19, 20, 22,
PUMA, 33 28, 37, 39, 41, 44, 45, 46, 47, 48, 51, 54,
pyrimidine, 51, 83 80, 83, 88, 89, 90, 95, 99, 101, 106, 112,
120, 123, 124, 128, 129, 141, 147
risk factors, 129
Q rituximab, 16, 17, 20, 28, 33, 35, 39, 44, 45,
46, 47, 49, 54, 62, 64, 78, 81, 92, 95, 96,
quantification, 105
99, 102, 113
quercetin, 35
RN-486, 22, 25
RNA, 80, 85, 90, 93, 111, 112, 126, 146
R rosiglitazone, 59
RP5264, 29
R788, 31, 52
radioisotope, 83
rash, 22 S
RB1, 119, 134
safety, 16, 19, 20, 21, 22, 24, 28, 29, 35, 36,
reactions, 34
37, 50, 51, 54, 90, 97
reactivity, 48
second generation, 21, 48, 82
receptor, 2, 5, 6, 7, 8, 9, 10, 15, 17, 18, 26,
secrete, 3
35, 36, 40, 44, 52, 53, 55, 56, 57, 60, 84,
secretion, 26
86, 94, 102, 104, 105, 121, 123, 127,
selectivity, 21, 23, 25, 29, 33, 47
137, 138, 139, 141, 142
senescence, 130
recognition, 139, 142
sensitivity, 25, 39, 41, 58, 118
recombination, 121, 124
sequencing, 85, 107, 117, 121, 125, 126,
recommendations, 100, 109, 137
127, 143
recovery, 69
serine, 51, 126
redistribution, 46
serum, 9, 41, 70, 72, 83, 84, 89, 101, 102,
regeneration, 69
103
relatives, 17
serum albumin, 70, 72
relevance, 71, 100, 101, 112, 129, 141, 146
sex, 4, 87, 88, 103
remission, 19, 33, 59, 81, 90
side effects, 37
replication, 117
signal transduction, 18, 32, 84, 105
repression, 41, 116
158 Index

signaling pathway, 15, 28, 32, 38, 43, 52, 78, 80, 81, 82, 83, 84, 85, 89, 90, 92, 93,
84, 121, 123, 127, 138, 139 95, 96, 98, 99, 101, 102, 103, 106, 107,
signalling, 53, 110, 138, 144 108, 109, 110, 111, 113, 114, 116, 117,
signals, 8, 31, 43, 49, 50, 53, 121 119, 120, 121, 122, 123, 124, 125, 126,
silica, 66 127, 128, 129, 134, 136, 137, 140, 144,
simvastatin, 39, 58, 59 147
siRNA, 66, 68, 70, 73, 75 survival rate, 78
SLE, 25, 26 survivors, 78
SNP, 117 susceptibility, 54
social problems, 2 Switzerland, 47, 51
sodium, 57 SYK Inhibition, 31
solid tumors, 30, 101 symptoms, 17
somatic mutations, 125 syndrome, 33, 59, 126
somnolence, 34 synthesis, 44, 58
Spebrutinib, 20, 21 systemic lupus erythematosus, 26
specialization, 62
spinal cord, 73
spinal cord injury, 73
T
spleen, 52, 53, 80, 121
T cell, 28, 30, 31, 36, 84, 105, 121
Spleen tyrosine kinase, 31, 52, 53
T cell receptor, 31
splenomegaly, 17
T lymphocytes, 10
squamous cell, 38, 57
tannins, 72
squamous cell carcinoma, 38, 57
target, 17, 18, 21, 22, 30, 31, 35, 36, 47, 48,
staging systems, 79
50, 52, 53, 55, 57, 59, 126, 139
stem cells, 57, 126
T-cell receptor, 84, 124
stereotyping, 124
TCR, 31, 104, 105
steroids, 20
techniques, 62, 118
stimulation, 8, 18, 32, 97, 118, 121, 125,
telangiectasia, 81, 86, 108
132, 135, 139
telomere, 108, 117
stratification, 44, 95, 99, 112, 147
teratogen, 59
stroke, 69
testing, 24, 37, 80, 114
stromal cells, 31
testosterone, 40
structure, 28, 37, 40, 42, 70, 71
TGR-1202, 28, 29, 50, 51
Styles, 114
therapeutic agents, 130
subgroups, 79, 94, 95, 123, 126, 129, 131,
therapeutic approaches, 9, 44
132, 145
therapeutic effect, 28
substitutions, 86
therapeutic goal, 91
substrate, 71
therapeutics, 100
suppression, 30, 43, 112, 126
therapy, 17, 19, 28, 33, 34, 36, 38, 41, 43,
surface modification, 42
44, 46, 59, 62, 63, 64, 77, 78, 79, 80, 81,
surveillance, 81
82, 83, 96, 99, 100, 111, 121, 123, 128,
survival, 2, 11, 16, 17, 19, 22, 27, 28, 29,
136, 139
30, 37, 43, 49, 50, 51, 52, 53, 55, 56, 58,
threonine, 126
Index 159

thrombin, 68 83, 85, 116, 119, 120, 121, 127, 129,


thrombocytopenia, 21, 31, 32, 34, 36, 80, 81 132, 133, 134, 135, 139, 144, 145, 147
thrombocytopenic purpura, 31, 40 tumor cells, 16, 33
time frame, 145 tumor growth, 34, 37, 52
tissue, 2, 10, 71 tumor necrosis factor, 55
titanium, 73 tumor progression, 2, 11, 63, 127
TLR, 111, 127 tumorigenesis, 110
TLR9, 55 turnover, 116
TNF, 25, 43 type 2 diabetes, 56
TNF-, 25 tyrosine, 15, 17, 18, 20, 21, 26, 31, 32, 45,
tonic, 27 47, 48, 49, 51, 52, 53, 70, 82, 84, 104,
tonsils, 130 105, 113, 121, 124, 127, 139, 141
toxicity, 16, 31, 32, 36, 42, 49, 66, 69, 70, Tyrosine, 45, 52, 86
71, 72, 75
toxicology, 16
TP53, 20, 28, 44, 46, 78, 81, 85, 86, 87, 89,
U
95, 96, 108, 109, 111, 120, 121, 125,
United States (USA), 4, 5, 74, 75, 111
126, 128, 129, 130, 137, 143, 145
updating, 50, 92, 131
TPA, 118, 132
training, 62, 129
transcription, 17, 37, 43, 44, 85, 86, 126 V
transcription factors, 37
transduction, 104, 105 validation, 129
transfection, 66 Valproic Acid, 16, 41, 59, 60
transformation, 81, 83, 94, 123, 125, 128 variables, 5, 99, 140, 146
translation, 57, 129 vascular endothelial growth factor, 52
transplant, 98 Venetoclax, 33, 54, 82, 97
transplantation, 82 viral vectors, 75
treatment, 15, 16, 17, 18, 20, 21, 22, 23, 24, VLA, 46
25, 27, 28, 30, 31, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 50, 51, 53, 55,
W
57, 69, 72, 78, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 92, 93, 95, 96, 98, 100, 102,
weight gain, 22
105, 107, 113, 115, 117, 118, 119, 120,
weight loss, 17
121, 122, 124, 128, 131, 140, 141, 145
World Health Organization (WHO), 4, 9,
trial, 19, 21, 23, 24, 25, 26, 27, 28, 30, 32,
132
35, 36, 46, 53, 56, 81, 92, 95, 96, 97,
100, 113, 139, 144
trisomy, 80, 81, 84, 86, 87, 94, 106, 110, X
116, 118, 120, 126, 129, 135, 136
tumor, 2, 3, 11, 16, 19, 21, 22, 29, 33, 34, xenografts, 21, 58
37, 39, 40, 42, 47, 52, 55, 56, 60, 63, 81,