Chronic lymphocytic leukemia (CLL) is derived from CD5+ B cells and is driven by genetic lesions and interactions with the microenvironment. CLL cells have abnormalities in apoptosis pathways like high Bcl-2 and FLIP expression that make them resistant to death signals. They also show chronic B-cell receptor signaling from tonic or antigen stimulation. The microenvironment protects CLL cells through cytokines and cell-cell contact with nurse-like cells and stromal cells. CLL cells harbor genetic changes like 13q14 deletions, trisomy 12, and mutations in NOTCH1 and SF3B1 that contribute to pathogenesis. Antigen stimulation may select for the restricted immunoglobulin repertoire in C
Part I - Anticipatory Grief: Experiencing grief before the loss has happened
Cll pathogenesis and targeted therapy
1. PATHOGENESIS OF CLL AND ITS
BASIS FOR TARGETED THERAPY
Presenter- Dr Abha Singh
Moderator- Dr Mrinalini Kotru
2. EPIDEMIOLOGY
CLL has average incidence of 5.17 persons per 100,000 in United
States representing 20% of all mature B cell neoplasms.
Accounts for 0.8 % of all cancers
Incidence is twice in males as compared to females
Generally, the neoplastic lymphocytes are of B cell origin
In less than 2 % cases, however, the neoplastic cells are of T cell origin
and are included in the category of T cell prolymphocytic leukemia.
5. ETIOLOGY
Mostly idiopathic
Familial cases
Cytogenetics- clonal chromosomal abnormalities are
detected in approximately 50% of CLL patients
Most common clonal abnormality is trisomy 12
Structural abnormalities of chromosome 13, 17 and 11
Patients with abnormal karyotypes have worse prognosis
6. CLINICAL FEATURES
Patients are more prone to viral or bacterial infections secondary to
impaired T-cell immunity or hypogammaglobinemia respectively.
Night sweats and fever (B symptoms) are uncommon and requires
prompt evaluation for complicating infectious disease.
80% of CLL patients have non tender LAP involving the cervical,
supraclavicular or axillary lymph nodes.
Lymphedema is rare.
7. LAB EVALUATION
Sustained monoclonal lymphocytosis greater than 5000/micro litre.
In B cell, clonality is confirmed by expression of kappa or lambda
light chain on surface membrane.
10-25% patients with CLL develop autoimmune haemolytic anemia,
with a positive direct Coombs test.
The marrow aspirates shows greater than 30% of the nucleated cells
as being lymphoid.
8. Continue lab evaluation…
Essential
Physical exam : attention to node bearing areas
including waldayers ring, and to the size of spleen
B symptoms
CBC, differential count, platelets
Comprehensive metabolic panel
9. OTHER INVESTIGATIONS
Quantitative immunoglobulins
Reticulocyte count, haptoglobin and DCT
Beta 2 microglobulin level
LDH
IGVH mutation status
Zap 70 molecular investigation
Radiological investigation before chemo-therapy (PET scan usually not required)
11. Points to be discussed
Origin of CLL
Possible drivers
Pathogenesis
Treatment
Targeted therapy
Approach to treatment
12.
13. ORIGIN OF CLL
B cells express CD5,CD19, CD23 and low levels of surface Ig
Recent gene expression profiling studies have confirmed that
CLL is probably derived from CD5 B cells similar to those
found in healthy individuals.
14. Cellular origin of CLL
SMZL
DLBCL
HCL,PLL
CLL( mutated)
MALT
CLL(Unmutated)
FL, DLBCL,
BURKITT’S
PLASMA CELL
DYSCRASIA
MANTLE CELL
HL
Naïve
B cell
Marginal zone
GC
MZ
15. Cellular origin of CLL : MUTATED VS
UNMUTATED
CLL( mutated)
CLL(Unmutated)
Naïve
B cell
Marginal zone
GC
MZ
ANTIGEN
MEMORY B CELL
16. CD5+ B cells
Gene expression studies on CLL cells revealed that leukemia
cells that express mutated or unmutated IGHVs share common
gene expression profile-suggesting common origin
On basis of several studies:
CLL cells with unmutated IGHVs are derived from mature CD5+
CD27- B cells
CLL cells with mutated IGHVs are derived from distinct, previously
unrecognised, subset of CD5+ CD27+ Post germinal centre B cells
with mutated IGHVs
18. DRIVER EVENTS IN CLL PATHOGENESIS
CLL
Mutations
TP53,
SF3B1,
NOTCH1,
ATM,
MYD88
Loss of
13q,
11q,17p
trisomy
12
Auto-
activation
BCR
Signaling
Apoptosis
microenvironment
Unknown
antigen
19. Mutations- at any stage of B cell development
HSCs bearing oncogenetic mutations may give rise to B cells with
modest growth/survival advantages
M-CLL- B cells with incurred immunoglobulin (Ig) somatic
mutation express mutated Ig heavy-chain variable-region genes
(IGHVs)
U-CLL- B cells that have not undergone Ig somatic mutations
express germ-line IGHVs.
The expansion of a CLL (or MBL) clone is associated with
de novo accumulation of additional genetic lesions,
interactions between the leukemic cells, accessory cells and antigens in the
leukemia microenvironment of lymphoid tissues .
21. The extrinsic apoptotic pathway plays a major role in apoptosis.
Six known death receptors (DRs) including tumor necrosis factor(TNF),
Fas(APO-1 or CD95), and DR4/DR5 (receptors for TNF-related apoptosis
induce ligand [TRAIL]).
Contain a cytosolic domain called the death domain, which recruits
adaptor proteins such as Fadd/Mort-1 to the receptor complex after
binding to ligand.
The recruiter adaptor protein has a death domain end and a death
effector domain (DED).
22. Once bound to the TNF receptor, the DED binds to caspases 8 and 10,
which then become activated by autoactivation .
One of these inhibitors is FLICE inhibitory protein (FLIP), which is a
homolog of procaspase 8 and contains two DED domains but lacks
proteolytic activity.
It been reported that CLL cells have high levels of FLIP expression,
rendering the cells resistant to DR-induced apoptosis.
23. Modulators of the Apoptotic Pathway in
CLL
The bcl-2 family consists of ∼20 members that can either promote or inhibit
apoptosis.
Located in the cell membrane, nuclear membrane, and mitochondrial membrane
Function by binding to other proteins or influencing cell permeability and the release
of cytochrome c from the mitochondria.
bax, bcl-xS, bak, and bad-promote apoptosis
bcl-2, bcl-xL, and mcl-1 - inhibit apoptosis
Another group (e.g., bag-1) can influence the activities of the other family members.
CLL cells have high bcl-2, bax, and bak levels but have low levels of bcl-xL and bad
24. p53 - induce apoptosis, and this occurs preferentially in tumor cells( a
feature that may explain the relative tumor specificity of anticancer
agents)
Mechanisms p53-induced apoptosis –
up-regulation in the expressions of the TRAIL DRs, DR4 and DR5,
increased expression of the proapoptotic Bcl-2 family members, Bax, Noxa, and Puma
p53 mutations are typically associated with deletions of the
second allele (deletion 17p13)
Mutations or p53 gene deletions are observed in 10 to 15% of
CLL patients
These abnormalities are associated with high lymphocyte counts, drug
resistance to anticancer agents in vitro and in vivo, and poor patient
survival
25. The ATM gene is located on chromosome 11q22–q23
Responsible for phosphorylation and activation of p53 after DNA damage
Approximately 30% of CLL patients have a mutation of ATM
These patients have a defect in cellular response to irradiation similar to that
observed in patients with a p53 mutation
Explains the drug resistance and poor clinical outcome in patients with an
ATM mutation
ATM mutations are seen in patients with an unmutated IgV gene, and these
patients are known to have a poor prognosis
26. Abnormalities in Cell Division
Both cyclins D2 and D3 are overexpressed in chronic lymphocytic leukemia cells,
but the retinoblastoma (Rb) protein is not phosphorylated, perhaps related to
overexpression of p27Kip1. CDK, cyclin dependent kinase.
29. Ligation of B cell receptor (BCR)
by antigen recruits kinases such
as spleen tyrosine kinase (SYK)
and the SRC kinase LYN that
phosphorylate immunoreceptor
tyrosine–based activation motifs
(ITAMs) on the cytoplasmic
domains of the immunoglobulin
(Ig) coreceptors CD79a and
CD79b.
Such phosphorylation recruits
and activates Bruton’s tyrosine
kinase (BTK) and
phosphatidylinositol 3-kinase
(PI3K), subsequently activating
many downstream targets,
including AKT/mTOR (mammalian
target of rapamycin), nuclear
factor κB (NF-κB), and
extracellular signal–regulated
kinase (ERK).
B cell signaling in chronic lymphocytic
leukemia
30. B cell signaling in chronic lymphocytic
leukemia
This signaling can be enhanced by ζ-associated protein of 70 kD (ZAP-
70). CD38, CD49d, CD44, and matrix metalloproteinase (MMP)-9 may
form a supramolecular complex with ZAP-70.
This complex can also recruit ZAP-70 to the plasma membrane, where
it can enhance BCR signaling.
31. BCR SIGNALING
CLL vs NORMAL B CELLS
CHARACTERISTIC CLL CELLS
sIg expression Low (higher in U-CLL)
CD79 expression Low
Response to antigen stimulation Variable (higher in U-CLL)
Calcium flux Variable/generally low
Syn/Lyn/Btk expression elevated
PI3K p110 delta expression normal
PI3K Kinase activity elevated
32. Cell Survival Factors in CLL
CLL cells interact with the microenvironment through activation of
their cell-surface receptors- main signal that promotes survival in CLL
cells.
CLL cells also produce cytokines-stimulate their own growth in an
autocrine or paracrine fashion while inhibiting survival of normal
lymphoid and marrow cells.
This latter effect can lead to the immunosuppression and
myelosuppression that typifies this disease.
33. The various cytokines and their receptors that contribute
to cell survival are
B-Cell Receptor (BCR)
Interleukins
Tumor Necrosis Factor-α (TNF-α)
Transforming Growth Factor-β (TGF-β)
Adhesion Molecule Receptors
BMCA, TAC1, and BAFF-R
Vascular Endothelial Growth Factor (VEGF) Receptor
CD40
35. CLL microenvironment
Chemokine receptors and adhesion molecules expressed by CLL cells
are critical for homing and retention of CLL cells within the tissue
compartments.
CLL cells receive prosurvival signals via contact with accessory stromal
cells in the leukemia microenvironment.
Nurselike cells express the chemokines CXCL12 and CXCL13, whereas
marrow stromal cells express predominantly CXCL12.
Nurselike cells and marrow stromal cells attract CLL cells via the G
protein–coupled chemokine receptors CXCR4 and CXCR5, which are
expressed at high levels on CLL cells.
36. CLL microenvironment
Nurselike cells also express the TNF family member BAFF and a
proliferation-inducing ligand, providing survival signals to CLL cells via
the corresponding receptors (BCMA, TACI, BAFF receptor).
Integrins, particularly very late antigen 4 integrins (CD49d), cooperate
with chemokine receptors in establishing cell– cell adhesion through
respective ligands on the stromal cells (vascular cell adhesion
molecule 1 and fibronectin).
Another important molecule involved in the interactions between
leukemia cells and their microenvironment is CD44
37. CD38, CD49d, CD44, and
matrix metalloproteinase
(MMP)-9 may form a
supramolecular complex with
ZAP-70.
This complex can also recruit
ZAP-70 to the plasma
membrane, where it can
enhance BCR signaling
Following binding to any or
all of these receptors by
ligands released by accessory
cells in the leukemia
microenvironment, AKT and
ERK undergo enhanced
activation.
CLL microenvironment
38. CLL microenvironment
CXCR4 can also directly interact
with CXCL12 to induce calcium
mobilization, activation of
PI3K/AKT, ERK, and serine
phosphorylation of signal
transducer and activator of
transcription 3 (STAT3).
Activation of Toll-like receptor
(TLR) can also enhance or
induce activation of NF-κB.
39. IMMUNOGLOBULIN REPERTOIRE
B cells are crucial to adaptive immune responses.
The probability that two independent B cell clones carry exactly the
same BCR is extremely low (e.g., less than 10−12).
CLL cells isolated from different patients often express similar, if not
identical, BCRs with common stereotypic features and/or structural
similarities.
IGHVs, such as IGHV1–69, IGHV4–34, and IGHV3–7, are used by CLL
cells at higher frequencies than those observed in normal B cells
Incidence of somatic hypermutation is not uniform among IGHVs in
CLL cells.
The marked restriction in the Ig gene repertoire of CLL cells highlights
the role played by one or more common self-or environmental
antigens in leukemic B cell selection
40. ANTIGENS THAT MAY PLAY A ROLE IN LEUKEMIA B
CELL SELECTION
Some of the Ig expressed in CLL can react with antigen expressed by
cells undergoing apoptosis, including cytoskeletal proteins
Some Ig react with non muscle myosin heavy chain IIA, which is
expressed on some apoptotic cells, namely myosin-exposed apoptotic
cells (MEACs).
Binding to MEACs is more commonly observed on CLL cells expressing
unmutated IGHVs than on CLL cells expressing mutated IGHVs.
In addition to self-antigen, several other microbial or virus-associated
antigens may contribute to the selection of the Ig expressed in CLL.
41. GENETIC ALTERATIONS IN CHRONIC
LYMPHOCYTIC LEUKEMIA
CLL cells commonly harbor deletions at 13q14, 11q22–q23, or 17p13 or
may have an extra copy of chromosome 12 (trisomy 12)
The advent of next-generation sequencing technologies, coupled with
gene copy-number analyses, have identified additional genetic lesions
in CLL, such as mutations in NOTCH1, SF3B1, and BIRC3
Such mutations could be used as potential therapeutic targets or as
biomarkers that can distinguish among patients who may have
disparate clinical outcomes
42. EVIDENCE OF BCR SIGNALING AS CLL
DRIVER
Strong association of somatic hypermutation status with overall survival
IgM rarely lost in CLL
sIgM levels have clinical implications
Specific and limited V gene use
Steriotyped BCRs
Evidence for autonomous BCR activity
Universal response to BCR pathway inhibition
Resistance associated with pathway mutation
43. Pattern of Clonal Evolution and Antigen
Dependence in CLL
GERMINAL
CENTER B
CELL
ANTIGEN
MUTANT SUB CLONE WITH
DYSREGULATED DRIVE BUT
STILL RESPONSIVE TO
ANTIGEN
MUTATIONS CONVERTING BCR TO
AUTONOMOUS ACTIVITY
44.
45. NOTCH1
Encodes a ligand-activated transcription factor
Regulates several downstream pathways that induce the
differentiation of hematopoietic progenitors immature T cells and
of mature B cells antibody-secreting cells
Activating mutations in NOTCH1 occur in ∼60% of T-lineage acute
lymphoblastic leukemias.
In CLL, activating NOTCH1 mutations have been detected in ∼10% of
newly diagnosed cases.
NOTCH1 mutations are also more frequent in CLL cell populations that
express unmutated IGHVs and that have trisomy 12.
46. NOTCH1 mutations
Restricted to the C-terminal PEST [proline (P), glutamate (E), serine (S),
and threonine (T)] domain
normally limits the intensity and duration of NOTCH1 signaling.
Removal of the PEST domain impairs the degradation of NOTCH1
allowing accumulation of the active form of NOTCH1.
One recurrent mutation (c.7544_7545delCT) accounts for ∼77% of all
NOTCH1 mutations in CLL-
Can be rapidly detected by a simple polymerase chain reaction–based
strategy- providing a potential approach for a first-level
screening of NOTCH1 alterations.
47. SF3B1
Encodes the splicing factor 3B sub-unit 1 (SF3B1)
Mutations in SF3B1 were observed in ∼10% of newly diagnosed CLL
cases and in ∼17% of cases with progressive, late-stage disease
requiring therapy.
SF3B1 mutations- acquired during clonal evolution,
the proportionate representation of sub-clones harboring SF3B1
mutations can increase over time, independently of cytoreductive
therapy.
SF3B1 regulates the alternative splicing program of genes controlling
cell cycle progression and apoptosis
Mutations in SF3B1 may enhance CLL cell
proliferation and/or survival
48. BIRC3
BIRC3 encodes the baculoviral inhibitor of apoptosis (IAP) repeat
Contains 3 protein (BIRC3)-
can inhibit apoptosis by binding to tumor necrosis factor (TNF) receptor–
associated factors 1 and 2 (TRAF1 and TRAF2)
possibly by interfering with activation of ICE-like proteases (caspases).
BIRC3 –
- act as an E3 ubiquitin–protein ligase that regulates nuclear factor
κB (NF-κB) signaling-
-acting to promote canonical NF-κB signaling while suppressing
constitutive activation of noncanonical NF-κB signaling.
Activation of canonical NFκB signaling can promote the
growth and survival of CLL cells in vitro and in vivo.
49. BIRC3 mutations in CLL are predicted to disrupt the C-terminal RING
domain, essential for proteasomal degradation of MAP3K14 by BIRC3
CLL cells harboring mutations in BIRC3 display constitutive NF-κB
activation appear less responsive to conventional chemotherapy
than CLL cells without such mutations
Inhibitors of NF-κB may have clinical activity in CLL-particularly in
cases harboring alterations in BIRC3.
50. MYD88
MYD88 encodes a critical adaptor molecule of the Toll-like receptor (TLR)
Mutations in MYD88 are observed in 3% to 10% of CLL cases at diagnosis
Also mutated in other B cell malignancies-
lymphoplasmacytic lymphoma
diffuse large B cell lymphoma (DLBCL)
marginal zone B cell lymphoma.
Mutations in other genes encoding proteins involved in the activation of TLR
signaling and NF-κB have also been observed in DLBCL.
In contrast to the mutations in NOTCH1 or SF3B1, mutations in MYD88 appear
to be present in the vast majority (if not all) cells within the CLL clone;
MYD88 appears to be an early driver mutation in at least a subset of CLL
cases.
51.
52.
53.
54. TREATMENT
In general, it is estimated that one third of CLL patients never require therapy
one third need treatment as soon as they are seen
one third have disease progression over the years and require therapy at some
point.
The indications to treat in CLL have been reviewed and are as follows:
1. Rai stage 0–II disease in patients who are symptomatic (weight loss of >10% body
weight in previous 6 months, extreme fatigue, night sweats or fevers of >100.5°F for >2
weeks without evidence of infection), have progressive anemia/thrombocytopenia or
lymphocytosis (>50% increase over 2 months or doubling time <6 months)
2. Rai stages III/IV to improve the hemoglobin level and/or platelet counts, although
asymptomatic patients can be monitored and treatment initiated when there is clear
evidence of disease progression
3. Bulky or progressive lymphadenopathy (masses >10 cm in diameter) or splenomegaly
(>6 cm below left costal margin)
4. AIHA/ITP
57. TREATMENT COMPONENTS
2. COMBINATION CHEMOTHERAPY
4. COMBINATIONS USING TARGETED THERAPY
1. SINGLE AGENTS
a) Cytostatic agents
b) Monoclonal antibodies
c) Agents targeting B-cell receptor (ibrutinib)
d) BCL-2 inhibitor
e) Immunomodulatory drugs
3. CHEMOIMMUNOTHEREPY
a) Rituximab
b) Ofatumumab
c) Alemtuzumab
d) Obinutuzumab
58.
59. TARGETING PATHOGENESIS
ACTIVATING TRANSLOCATIONS: if they affect a targetable gene
BCR, cyclin D1, MYC
ACTIVATING MUTATIONS: if they affect a targetable gene and are true
disease drivers
Kinases
CONSTITUTIVELY OR ABERRANTLY ACTIVATED PATHWAYS THAT ARE NOT
MUTATED- BCR signaling
60.
61. TARGETED THERAPY
For patients who have stopped responding to treatment new drug
combination may be effective option
Based on phase 3 clinical trials venetoclax and rituximab reduced the risk of
cancer progression by more than 80%
We are entering an era in which CLL treatment is not one size fits all but
instead tailored to the biology of each person’s disease, depending on
markers and genetic mutations.
Clinicians now recommend that all patients with CLL to get some sort of
genetic screening.
62. TARGETED THERAPY (Cont.)
First class drug works by interfering with B cell receptors.
These receptors are crucial for proper proliferation- if they go rogue- turn
cancerous.
New drugs take advantage of this by jamming this signalling system, creating
agents that bind to receptors and block those that allow cancerous cells to
multiply or by interfering with kinases.
63. IBRUTINIB
First-in-class Bruton tyrosine kinase (BTK) inhibitor
Blocks B cell receptor (BCR) signaling, a key pathway for CLL cell
survival and proliferation.
Has potent activity even in high-risk groups such as previously treated
CLL or CLL with TP53 aberrations
Approved for all CLL patients based on improved progression free
survival in treatment-naive disease and a favorable safety profile
Increasingly used as monotherapy or tested in combination regimens.
65. IMBRUVICA
FDA approved
Blocks Bruton’s tyrosine kinase (BTK), and several other kinases
For patients with 17p genetic mutation this is first line treatment
66. ZYEDELIG( IDEALASIB)
FDA approved
Blocks phosphoinositide 3 kinase (PI3K)
Most affective when combine with Rituximab
68. Others
GAZYVA( obinutuzumab)- CD 20 monoclonal antibody
Approved as first line treatment in CLL in combination with the chemotherapy
drug chlorambucil
ARZERRA (ofatumumab)- has been approved in later lines of therapy
