Session
agenda and rationale:
·
Purpose: Review each of the myeloproliferative diseases and
address issues related to abnormal signaling and cytokine
mediated events
·
Challenge: To collectively identify what is known about signaling
in these diseases and where gaps are in our knowledge
· Questions to address for each disease
- #1: What is the hard evidence that abnormal
signaling contributes to abnormal pathophysiology
in myeloproliferative and mast cell disorders?
* MPDs are considered
diseases of abnormal signaling, but for each, ask, "Does
abnormal signaling contribute?" - from literature and participant
insight
* If no evidence, how
can it be established that abnormal signaling contributes?
- #2: If there is evidence that abnormal signaling
contributes, how can the nature of these
signaling abnormalities be established?
- #3: What are the therapeutic implications?
- #4: What are the resources needed to answer
these questions in these rare disorders?
* How can clinical resources
be pooled to evaluate signaling pathways?
* Identification of
compounds and pathways in terms of therapeutic implications?
MAST CELL
PROLIFERATIVE DISORDERS (MASTOCYTOSIS) (DR. KAUFMANN)
·
Evidence of abnormal pathophysiology
- Strongest evidence of abnormality
* c-kit mutations occur
in malignant mast cell lines
* c-kit mutations also
occur in human clinical samples
* If mutations are introduced
artificially into cell lines, result is ligand independent
signaling through the c-kit receptor
- 2 types of c-kit mutations
* Juxtamembrane mutation
(regulatory mutation)
* Activation loop or
enzyme-pocket reactivation mutation
·
Establishing the nature of signaling abnormalities
- Downstream of these mutations
* Activated c-kit activates
PI3-kinase AKT survival pathway and SRC pathway
* AKT and SRC are well
known anti-apoptotic pro-proliferative pathways
* Supporting evidence
in models and mast cells from knock-out mice where activated
c-kit is anti-apoptotic and pro-proliferative
·
Therapeutic implications
- STI571 and similar compounds - inhibitor of
c-kit
- Recent publication - activation loop(D816Y)
c-kit mutants are resistant to STI571
- Memorial investigators divide c-kit into mutants
in regulatory domain (sensitive to STI571
and mutants in activation loop (enzyme-active site; resistant
to STI571)
- Activation loop c-kit mutation similar to
STI571-resistant CML and ALL at relapse - pharmacology
of STI571 (binds to inactive conformation of BCR/ABL and c-kit)
- Implications
* not every c-kit mutation
is same
* need other c-kit inhibitors,
especially to inhibit D816Y
·
Resources needed to answer questions
- How to screen other inhibitors/mutations and
bring to clinical trials for investigation?
(In cell lines? In transfected cells? In clinical material?)
·
Discussion
- What to do now that the lesion is known?
- Are c-kit mutations found in 98% of mast cell
disorders or only a smaller percentage
- Should the possibility of other mutations in the same pathways
be assessed?
- Similar seminoma study (Dr. Murgo) - (AACR
abstract/Oregon Health & Sciences
Univ.)
* patients with seminoma
have mutations similar to mast cell disease
* evaluated large panel
of specimens of seminoma and identified mutation
* transfected cells
with mutation and tested cells for sensitivity for STI571
* data in seminoma similar
to mast cell disease - majority of activating mutations
only occurred in 25%; majority of mutations identified were relatively
resistant
to STI571
* benefit - model screens
relatively large number of samples, identifies mutations,
and then evaluates for sensitivity to drugs like STI571
- Technique at Memorial (Dr. Kaufmann) - tested
c-kit mutations by placing in COS
cells and observe for inhibited autophosphorylation
- Another technique (Dr. Dunbar) - place mutations
in factor-dependent hematopoietic
cell line (e.g., BAF3) - observe to see if inhibitor prevents
factor- independent growth
(similar study done by Dr. Gilliland
in CMML with tyrosine-kinase activating mutations)
- C-kit mutations in BAF3 cells were not
inhibited by STI571
- Are any other inhibitors in development active
against these mutations?
* Dr. Gilliland -uncertain
activity against D816Y
· What
percentage of mastocytosis patients harbor the c-kit mutation?
· Assumption
that c-kit present in mastocytosis close to 100%
· Of those
with c-kit, what proportion have D816Y? majority of cases?
· Need
compound that inhibits D816Y to treat mastocytosis
·
Mutation is identified - How to find inhibitors to target D816Y?
- Screening with other inhibitors for D816Y
inhibition? (Dr. Gilliland)
- Compounds from Milennium and Sugen which hit
wild-type kit and GIST mutations
might be worth testing against D816Y
CHRONIC
MYELOMONOCYTIC LEUKEMIA (CMML) (DR. DUNBAR)
·
Evidence of abnormal pathophysiology
- Initially classified as a myelodysplastic
syndrome, but WHO recently reclassified CMML
as an overlap syndrome (borderline/MPD)
- Review of recent large study -
* Measured median survival
- median survival 12 months
* Analyzed prognostic
score (by MD Anderson)
* No clear separation
of cases with or without dysplastic changes in marrow pathology
(1/3 dysplastic; 2/3 proliferative)
* Traditional prognostic
indicators - monocytosis, rule out BCR/ABL, <20% blasts
* Most had informative
cytogenetics, but none had cytogenetics suggestive of PDGF
receptor or fusion oncogene (unlike mastocytosis with mutations
present
in majority)
* Identification by
cytogenetics is much smaller group
- Possible mutations - c-kit and PDGF-beta receptor,
others possible
- Other Potential Abnormal Signaling Pathways
in CMML
* C-FMS mutations in
MDS/CMML
* Patient mutations
at 301 or 969 - tyrosine kinase activation
* 110 pts with MDS/AML
* 12.7% of 969 mutations,
1.8% with 301 mutations
* 20% of patients with
CMML
* Higher frequency of
transformation to AML in follow-up study
·
Establishing the nature of signaling abnormalities
- Fusion oncogene in CMML identified with translocated
TEL to PDGF-beta receptor (Dr. Gilliland)
(similar activating mutation of tyrosine kinase with BCR/ABL)
* PDGF-beta receptor
present in CMML : fusion partners : TEL, HIP, Rabaptin dimerize
and activate the PDGF-beta receptor
* Analysis ongoing -
what partner fusion proteins are involved? do partner fusion proteins
confer special characteristics for patients? - little clinical
data
- CMML case (Dr. Dunbar) - cloned fusion protein
from patient with non-dysplastic, proliferative,
undifferentiated CMML (BCR/ABL negative, 5:17 translocation)
* compared sensitivity
of fusion oncogene to STI571 vs. BCR/ABL - fusion oncogene
more highly sensitive than BCR/ABL
* case history - early
allogeneic transplant - cytogenetically negative 12 months -
transcripts began to reoccur at 15 months with GVHD - STI571 treatment
given and transcripts disappeared
- sustained remission
- HIP/PDGF-R translocation (Dr. Melo): - one
typical TEL/PDGF mutation; one with
unknown partner; no transplant - complete cytogenetic remission
>14 months with STI571
- Where CMML lacks other therapeutic alternatives,
provide justification to develop Phase
II trial to evaluate response to STI571
* One Phase II
trial currently in myelofibrosis includes CMML
* Could STI571
work in the absence of PDGF receptor fusion? (Dr. Gilliland)
* Could an activated
receptor fail to be detected by conventional cytogenetics? (Dr.
Dunbar) - suggest a large-scale FISH trial or other screening
* Would other
activated tyrosine kinases cause the same phenotype? Is this the
default pathway in a primitive myeloid progenitor that causes
an activating mutation
in a tyrosine kinase?
* Mayo trial using
STI571 in CMML was closed prematurely(Dr. Kaufmann)
due
to fatal splenic rupture. These patients did not have translocation,
no response
to STI571
·
Caution against opening a trial unless there was evidence for
this translocation
·
Heterogeneity in signaling may produce syndromes looking exactly
the same
·
Potential causal relationship with STI571 - splenic rupture not
seen in patients
who did not receive STI571
·
Questions -
·
Were patients on erythropoietin? (Dr. Spivak) - Experience with
splenic rupture
in CMML patient with erythropoietin - spleen can enlarge rapidly
*
Getting requests to use STI571 in patients without cytogenetic
abnormality (Dr.
Gilliland) - currently no recommendation - offering to sequence
the PDGF, kit,
and SH3 domain of ABL as known targets
· Clinical
response in case at Baylor where activating mutation not identified
after
sequencing the entire receptor and SH3
· Screen
thoroughly and use caution - should not preclude treatment with
tyrosine
kinase inhibitors in patients without known activating mutations
(by cytogenetics
or point mutations) - some remarkable responses seen
· HES
study has shown good response in 5 of 5 patients - signaling defect
is not
known but therapy is successful (Lancet, 2002)
- Publication on C-FMS in CMML in early 1990s
- was this confirmed? (Dr. Dunbar)
* C-FMS studies
used allele-specific priming ; subsequent studies could not identify
mutation with DNA sequencing (Dr. Gilliland)
-
Is peripheral blood or marrow being sequenced? Differences? (Dr.
Magnuson)
* Possible lineage-specific
expansion (but broad-spectrum myeloid expansion in
CMML)
* Is it sufficient to
sequence bone marrow?
* No known evidence
for disparity between bone marrow versus blood in sequencing
results (Dr. Gilliland)
·
peripheral blood is reasonable target for sequencing
·
most patients have leukocytosis and monocytosis in peripheral
blood
·
strongly advocate sequencing all exons of c- kit, PDGF-beta receptor,
PDGF-alpha
receptor, and SH3 of ABL
* Is there evidence
that all myeloid lineages are clonal in CMML - in the megakaryocytic
line? (Dr. Prchal)
·
some cytogenetic evidence - published study on MDS patients that
included some
CMML found that megakaryocytes were involved (Dr. Gilliland)
·
question of whether cells are part of the clone - compared to
hypereosinophilic
syndromes where investigators asked if eosinophils themselves
were part of the clone (Dr. Dunbar)
·
similar issue in array technology for expression of proteomics
where it is sometimes
difficult to identify the most relevant cell population related
to expression
- CMML is similar problem due to wide heterogeneity of expansion
in different compartments
- Another Potential Screening Alternative for
STI571 Trials (Dr. Melo) - Novartis "basket
protocol" in Europe screening candidates who potentially
benefit from STI571 therapy
* Trial includes any
malignancy where kit or PDGF receptor mutation is suspected
* Patients must have
positive confirmation of a mutation sensitive to STI571 or, without
a mutation, have confirmation for sensitivity of cells to STI571
in vitro
* Hammersmith has two
assays for cells culture to treat cells in vitro with STI571
to look for response as an indication that there will be in vivo
response
* Clinical responses
parallel positive in vitro responses, particularly in patients
who appear
to have potential PDGF receptor involvement, but no confirmation
* Results could provide
definitive evidence that other receptors are STI-sensitive -
also similar indications from HES patient data (Dr. Gilliland)
- Clarify concept behind "basket protocol"
- Is STI571 targeting fibrosis at the PDGF
receptor expressed as a wild-type receptor in stromal elements?
(Dr. Gilliland)
* Interest/Consideration
from trial examples (Dr. Gilliland)
·
Over-expression of wild-type kit or PDGF receptors do not render
the cells dependent
on those receptors for survival
·
Only cells that have activating mutations seem to be addicted
to the presence
of the receptor - why?
·
Why are cells induced into apoptotic cell death when the PDGF
receptor is turned
off? All other receptors are presumably normal (in the cytokines,
in the
milieu, etc.)?
·
Support for in vivo experiment - some responses could be seen
in patients only
over-expressing the wild type receptor vs. excluding patients
just based on
sequence analysis
- Regarding STI571 targeting fibrosis - Is an
ongoing trial examining an anti-PDGF agent
stopping fibrosis? (Dr. Spivak)
* Mouse model with over-expression
of PDGF - fibrosis not part of that syndrome
* No known evidence
that PDGF is involved in fibrosis - is there new evidence?
* Monocytes generally
thought to be involved in creating fibrosis in MPDs
* STI571 may hit another
receptor which affects fibrosis, but PDGF unlikely
* New phase II STI571
study for myeloid metaplasia and CMML (University of Chicago,
Dr. Gilliland) - primary endpoints is clinical response, changes
in fibrosis
will also be an endpoint
* Study does not directly
examine relationship of PDGF expression and fibrosis
AGNOGENIC
MYELOFIBROSIS/MYELOID METAPLASIA (MMM) (DR. KAUFMANN)
·
Identify the questions for myelofibrosis and signaling based on
earlier commentary
- controversy as to the potential role of PDGF
- or no evidence of a role for PDGF?
·
Evidence of abnormal pathophysiology
- Proposed pathogenesis of myelofibrosis
* Signaling disorder involving PDGF,
TGF-beta, basic fibroblast growth factor (bFGF)
* Possible clonal megakaryocyte
proliferation or clonal monocyte/histiocyte proliferation
- generates cytokine "storm" of neoangiogenesis, osteosclerosis
and reactive fibrosis
- Fibroblasts are polyclonal; myeloid precursors
are clonal
- Consequently bone and fibroblast reactions
are reactive
* PDGF and TGF-beta
are fibrogenic and both produced by platelets
* possible that megakaryocytes
and platelets are contributing to fibrosis
* French article published
showing elevated platelet levels of PDGF and TGF- beta
in myelofibrosis
- Other Molecules Implicated
* Forced thrombopoietin
(TPO) over-expression in bone marrow cells
·
Leads to initial elevation of platelets and neutrophils
·
Marrow fibrosis, osteosclerosis, and extramedullary hematopoiesis
follow
·
In animal model, initially looks like ET and ultimately looks
like post-ET fibrosis
with elevated PDGF and TGF-beta
* Why are fibrogenic
cytokine levels elevated? - Are they truly elevated?
* One report of response
to TGF-beta was biphasic - high levels of TGF-beta are thought
to inhibit marrow fibroblasts rather than stimulate
* Is there possible
abnormal signaling by fibrogenic cytokines?
·
Disorder where there is guilt by association
·
How to establish whether this is a signaling disorder?
·
How to establish which cytokines are involved?
·
How to target those cytokines? - not to just blindly inhibit PDGF,
TGF-beta or
TNF-alpha (also implicated)
- Classic patients with myelofibrosis often
have end-stage disease upon presentation
with degradation of many pathways (Dr. Spivak)
* Present with massive spleen
and highly fibrotic bone marrow
* Cannot identify where disease
started
* Need to develop ability
to diagnose patients in earlier stage to identify markers
· Pathology
data from Dr. Thiele - recognition of "hyperproliferative
stage" of idiopathic
myelofibrosis - namely seeing it before significant fibrosis
- Guilt by association caused by other processes
following initial disease process
- Association path, not causality
* See PDGF, TGF-beta and TNF-alpha
- these can cause stimulated fibroblasts - assume
causality - Causality not established
* Model evidence
· Non-Skid mouse
model - fibrosis not possible because of a monocyte defect
· Skid mouse
- can create fibrogenic effect in presence of monocytes
· Recent data
showing that monocytes are central to the process
· Data shows
TGF-beta may be central to the process
* Why do some patients with idiopathic
myelofibrosis and other myeloproliferative disorders
get fibrosis?
· Cases where
patients have millions of platelets for decades without any fibrosis
vs. published cases of ITP and lupus patients with severe fibrosis
* Thrombopoietin data fascinating
- can create reversible fibrosis with TPO - one mechanism,
are there others? Some other models have not seen elevated TGF- beta
or PDGF-beta with elevated TPO
- Focus on fibrosis not useful for abnormal
signaling and cytokines (Dr. Dunbar)
* fibrosis too variable, an end-stage
clinical problem
* underlying problems - myeloproliferation
and clonal hematopoiesis
* must identify the molecular defects
first; then approach them therapeutically
* obstacles
· no clear animal
model
· difficult to
get cytogenetics because many patients are not aspirable
·
Refocus discussion (Dr. Kaufmann)
- Is there agreement that idiopathic myelofibrosis
(or MMM) is a clonal disorders? If
agreed, what is the nature of that clone? How does it give rise
to fibrosis?
* Evidence of clonal disorder -
clonality shown using classical informative female for
isoenzymes of G6PD (Dr. Spivak)
* Possibly polyclonal myelopoiesis,
but that is probably secondary myelofibrosis (Dr.
Prchal)
* Few patients analyzed - clearly
evidence for some clonal myelopoiesis but also possibility
for polyclonal myelopoiesis in larger numbers (Dr. Gilliland)
* Animal models support possibility
that fibrosis is variable in apparently the same
phenotypic disease - disconnect CMML phenotypes from fibrosis
(Dr. Gilliland)
· most phenotypes
have high levels of megakaryocytes like TPO over- expression
· all result
in megakaryopoiesis and fibrosis
· animal model
where BID (a BH3 only pro-apoptotic gene) is deleted - result
is a
disease that looks exactly like CMML but the animals never develop
fibrosis
* Re-emphasize - not enough trials
to tell if all patients have the same clonal level -
might some patients have clones that arise in more mature states?
(Dr. Spivak)
* No consistent cytogenetic abnormality
or chromosome abnormalities seen in these
patients - some evidence for 12q, 5q, etc. (Dr. Kaufmann)
·
Resources needed to answer questions
- Possible recommendations (Dr. Kaufmann)
* Cooperative Groups or form large
consortium to start collection of material
· Should criteria
include confirmation of clonality for material to be allowed to
go into
the bank to be studied?
· What should
the material be studied for?
- Using 5 informative markers, we have success
in 90% of females; study platelets and
granulocytes using a transcription clonal assay (Dr. Prchal)
- Base Banking Criteria on Clinical Diagnostic
Criteria (Dr. Spivak)
* Avoid targeting one chromosomal
abnormality since this disease has vast heterogeneity
* First identify clinical diagnostic
criteria for idiopathic myelofibrosis
· #1 - splenomegaly
- whether presenting with a massive spleen in fibrosis or with
no discernible symptoms which in a few years develops fibrosis
· 95% of patients have
splenomegaly - would the other 5% that present with fibrosis
without splenomegaly, have the same chromosomal abnormality?
· #2 - fibrosis present
in the marrow
· #3 - blood count
abnormality
· variable - in red
cells, white cells or platelets
· generally anemic;
generally have leukocytosis or thrombocytosis
· maybe pancytopenia
* Other criteria available include an
Italian consensus group
* Need to agree on clinical criteria before
we start banking material - need to clearly
characterize the disease
- Propose creating an inclusive national registry
(Dr. Spivak)
* The myeloproliferative disorders
are rare, and myelofibrosis the least common
* NCI has now recognized MPDs as
neoplasms because they are clonal
* Need to raise general awareness
to learn more about the disease - clarify diagnostic
criteria, identify early disease stages
* Need a large cooperative group
before discussing what to measure
- Suggest collecting bulk specimens using very
broad criteria (Dr. Gilliland)
* Be more inclusive than exclusive
* Only low numbers of chromosomal
translocations
* Use bank to determine disease
groups - use broadest criteria, do clonal analysis,
and analyze to separate into different groups
* Regardless of pathophysiology,
gain knowledge on disease by banking samples;
by getting patients registered on clinical protocols to get those
samples
* Genetically challenging - known
cytogenetics are almost exclusively deletions
* Not enough of material to even
do careful deletional mapping on 5q
* Use bank to apply modern strategies
like high through-put sequencing of kinases
- doable using chip based strategies
* Critical to characterize specimens
to know they are clonally derived - often difficult
aspiration, not always sure if cells are clonally derived (Dr.
Gilliland)
- In bank, include a patient's therapeutic responses
to agents - even if the reasons for
response are unclear, this information may help to define the
disease Is this group proposing
that a bank be established and/or funded through NCI? Guidance
for the plenary session? (Dr. Kaufmann)
* Recommendation should address
both NCI and NHLBI (Dr. Wu)
* Support a bank, but need to agree
on what tissues to bank (Dr. Prchal)
* Would fresh samples be needed
for clonal analyses discussed? (Dr. Dunbar)
* Samples can be frozen if platelets
and granulocytes are isolated, or monoclonal cells
(Dr. Prchal)
- Banking vs. clinical trial: Experience from
GIST (Dr. Murgo)
* GIST - little known before successful
treatment identified (STI571) - raised interest
among researchers, clinicians and lay - internet promoted communication
among patients
* Banking initiated within context
of Intergroup clinical trial (SWOG led; CALGB coordinated
correlative studies and banking) - intense correlative study to
collect samples
and to look at types of correlates to try to identify characteristics
which were associated with outcome
* Key - needed a trial as basis
by which samples were collected
* Reason STI571 tested in GIST -
kit mutations were known - rationale for testing established
because biology was done beforehand - for MPDs, need to get enough
samples to understand the biology of idiopathic fibrosis (Dr.
Kaufmann)
* Wild type kit also found to respond
to STI571 in proportion of patients - not appreciated
before trial - similar discussions occurring for wild type PDGF
- but the major interest
in GIST came about after this study (Dr. Murgo)
- Establishing a national bank - not through
clinical trials (Dr. Spivak)
* Successful treatment for MF may
be a long time coming
* Need a mechanism to set up a tissue
bank (maybe through RFP/R3)
* Bank samples must be made available
to other investigators (for clonality studies,
sequencing, activating mutations)
* Recent clinical trials have been
unsuccessful (Enbril, STI571)
* Need tissue to go back to basic
science to find better options
* Small patient population - need
big Cooperative Group
· Start a tissue
repository to spawn scientist interest to study these diseases
· Great ideas
but few patients, need access to tissue
· Ideas in the
milieu - data available that CD34 positive cell population is
greatly expanded
in the peripheral blood - easy to obtain - place to start
* Rarity of patients - PV study
group needed 20 institutions to get 431 patients enrolled
on their pivotal trial - in similar time frame, Mt. Sinai had
200 PV patients but
only about 40 myelofibrosis patients - MF patients had great diversity
in presentation (Dr. Fruchtman)
* Need national attempt to get Cooperative
Group studies with enough patients
· Recognize genetic
and phenotypic diversity to all these patients
· Weakness of
recent trials - to draw conclusions about efficacy of novel agents
with
only 10 patients on trial
· Take national
approach to acquire patients for both banking and therapeutic
trials
- Need to collect specimens prior to therapy
and after response to therapy (Dr. Zeldis)
* Best responses in current trials
have had 20% to 30% response
* What happened to the other 70%?
* If therapeutic response helps
to define the disease, do the rest have another disease?
- Interaction with patient support group organizations
may prove helpful (Dr. Prchal)
* Patients are interested - valuable;
anxious to interact; willing to participate in tissue
banking and studies
* For the very rare disorders, even a
Cooperative Groups may be unsuccessful - need
broader participation - interact through these patient organizations
* The Myelofibrosis Foundation may be
able to help in setting up the bank (Dr. Kaufmann)
- Is successful accrual to a cooperative tissue
bank a reasonable expectation in the
absence of a clinical trial? (Dr. Gilliland)
* Considerable effort via trials
to collect samples at independent institution
* Is there willingness to send those
samples to a central bank and have them accessible
through committees?
* Would it work better under the
auspices of a trial where correlative science studies
could be more easily facilitated?
- Create the bank outside of trials (Dr. Spivak)
* Trials are attractive because
they draw patients interested in being treated - little
definitive knowledge to apply to a trial
* Trial design usually requires
narrowing patient selection for treatment-specific goals,
need the bank to represent widely inclusive disease population
- these diseases have
only some clinical definitions; heterogeneity
* Tissue bank independently to start
- use trials to get more patients if necessary
* Bank is essential to attract basic
scientists - meet their needs
· Need access
to tissue repeatedly
· Need funding
to support these studies
* Need government support (e.g.,
identify that MPDs are more frequent than CML,
intention to improve quality of life for these patients)
- Would institutions be willing to contribute
collected samples to a central bank? (Dr.
Gilliland)
* Dr. Spivak supported this for
his samples - benefits to contributing samples to a national
bank is receiving other samples back - works both ways - win-win
- Logistical point on patient consent and IRBs
(Dr. Prchal)
* More regulatory controls involved
to do research now than 5-10 years ago
* Interacted initially with a physician
in Ohio to identify a patient, IRB regulations required
all subsequent communication be made only with the patient directly
- obtained consent
but was not permitted to contact the physician for clinical information
* Difficult to get approval for
interaction with other institutions even for rare disorders
- each institution would need IRB approval from their own IRB
as well as coordinating
IRB
* HIPAA regulations require that
samples be stripped of personal identifiers, but can
include some clinical information (not traceable back to the patient)
without problems for
IRB approval
- NHLBI has tissue bank core resources where
large number of tissues are banked from
trials which NHLBI is running - subcontractor relationship - allows
for storage and shipment of specimens
(Dr. Wu)
* Might be feasible to design a
collaboration between NHLBI and NCI for accruing patients
with storage and shipment of specimens through NHLBI
* Banked specimen data needs to
include response to various therapies - would need
to collect patient specimen consent prospectively so research
can be done on specimens
to look at responses to specific treatments without requiring
additional consent on those samples - need to plan this ahead
(Dr. Kaufmann)
POLYCYTHEMIA
VERA (PV) (DR. DUNBAR)
·
Evidence of abnormal pathophysiology
- Clonally characterized in erythrocytes, granulocytes
and platelets
- Erythroprogenitors are hypersensitive to erythropoietin
(EPO) and cytokines present in serum
- In acquired PV, no evidence that binding or
the EPO receptor itself are abnormal
- Only mutations in EPO receptor found are in
familial erythrocytosis, but not in myeloproliferative
type PV
·
Establishing the nature of signaling abnormalities
- Focus of Dr. Spivak's research - c-MPL/thrombopoietic
(TPO) interactions
* Overexpression of TPO resulting
in fibrosis and extramedullary hematopoiesis
* Ectopic c-MPL can cause erythroblastosis
in certain models - published data
· Abnormalities
in protein phosphorylation in response to TPO in PV
· Decreased c-MPL
expression in platelets
· Are there any
new theories or new data on how this relates to pathophysiology?
How could this be translated into new therapies?
* Mutant EPO - potential treatment
for primary and secondary erythrocytosis
· R301 erythropoietin:
partial agonist/antagonist
· Inhibits proliferation
but allows differentiation in cell line models
· Mechanisms
not completely clear - animal model testing
* PV and c-MPL - impaired glycosylation
not understood, but present in CD34- positive
cells - these cells have growth advantage over normal cells in
vitro - sensitive to
IL-3 unlike normal CD34 positive cells (Dr. Spivak)
· In signal transduction,
difficult to find tyrosine phosphorylation in these cells
· Not finished
with studies on phosphatase inhibitors
· Occasional
single abnormal protein phosphorylated in some patients
· In CD34-positive
cells, hypo-tyrosine phosphorylation present; occasionally a very
specific, unidentified protein is phosphorylated
· Signal transduction
abnormalities present
· Taken under
glycosylated MPL and express it in an IL-3 dependent cell line
and get
same signal transduction abnormalities
· Reproducible
- tracks with disease, but not able to determine if related to
pathophysiology
of disease
* PV cells are sensitive to all
the growth factors that control normal hematopoiesis
- Any attempt to treat with an anti-EPO might be symptomatic treatment
but does not target the heart of the disease - agreed with Dr.
Prchal and others
* What does it mean in terms of
signaling pathways if patients are hypersensitive to
EPO or IGF1? - Is the defect then downstream (at global PI3 kinase
or others)? (Dr. Kaufmann)
·
Compensatory effect (Dr. Spivak)
·
When one receptor is not working, other receptors take over its
place
·
Some inhibitory pathways not up-regulated normally because receptor
signal is
not as strong as it might otherwise be
·
Similar to idiopathic myelofibrosis - intriguing clues but no
smoking gun
·
Same evidence in cytogenetics - evidence of some suppressor genes
·
Multiple things going on
·
Abnormalities appear to be downstream of primary lesion but primary
event not
clearly identified (Dr. Prchal)
Cytogenetic
approach looked at:
· both clonal and
polyclonal cells from the same individuals
· various T-cells
and myeloid cells
· did comparative
genome wide screening
· also look for
heterozygosity
· due to time requirement
only looked at 10 patients to date
· 4 patients had
heterozygosity in chromosomal region already extensively sequenced,
but found no mutation
· Data on this
same region - some gene messengers were up-regulated (including
NIFB, master regulator of many genes)
· Logically, primary
event may control many abnormalities
* Follow-up on EPO (Dr. Prchal)
· If bulk EPO and
EPO receptor antibodies are added to erythroid-independent clone
in serum, normal bulk erythropoiesis but not PV erythropoiesis
occurs
· Logically, an
EPO antagonist would not be beneficial
· If several genes
are activated by same abnormality - EPO agonist would also fail
to be beneficial
* Heterogeneity of disease identified
by PV Study Group criteria (Dr. Spivak)
· Criteria
does not define all patients at start of illness
· Some only
erythrocytosis, some erythrocytosis and thrombocytosis, small
minority
with trilineage hyperplasia
· Heterogeneous
patient history - some long-term without problems, some with abbreviated
clinical course, some develop myelofibrosis, some present with
myelofibrosis
with increasing red cell mass
· Disease not monolithic
- speaks for acquiring tissue from patients with particular
characteristics
* Abnormality with loss of 9p
looks to be very important (Dr. Spivak)
· Found chromosomal
lesions and non-chromosomal lesions (Dr. Prchal)
· Found loss of
heterozygosity which spanned wide region (40 centiMorgans); not
chromosomally visible
* Other possibilities which
might coincide with finding compensatory mechanisms
for a primary genetic defect (Dr. Gilliland)
· Haploid gene
dosage could be the only abnormality or a contributor
· Possible strategy
to look at haploid gene dosage using modeling in Zebra fish
· How extensively
has phosphatase deficiency been examined?
· Usual popular
phosphatases (7 or 8) were cloned out - all found to be normal
· Found 1 phosphatase,
which was not recognized initially, that was hyper- expressed
in PV - megakaryocyte phosphatase (megPTP)
· Sequence is normal,
but megPTP is hyperactive
· May contribute
to hematopoiesis again, but not the primary defect
· Others available
could be looked at, but usual methods to choose tyrosine phosphatases
and cloning on that basis have not revealed anything to date
JUVENILE
MYELOMONOCYTIC LEUKEMIA (JMML) (DR. EMMANUEL)
·
Evidence of abnormal pathophysiology
- More known about disease signaling
- BCR/ABL negative myelomonocytic leukemia (MML)
in children
- Most present at less than 2 years of age with
anemia hemorrhage, skin rash, lymphadenopathy
- Heterogeneous clinical course
- Clinical and pathophysiology overlap
* monosomy-7 syndrome hyper-responsive
to GM-CSF
* work by Dr. Kevin Shannon identifying
genetic abnormalities from work on neurofibromatosis
·
Establishing the nature of signaling abnormalities
- Activating ras mutations found in 30%, specific
to those without neurofibromatosis
- NF1 - loss of normal NF1 allele in patients
with JMML and neurofibromatosis
* NF1 protein, neurofibronin (GTPase)
that down-regulates ras-GTP
* In normal situation, GM-CSF and
other signaling pathways maintain ras-GTP in inactive
state
* In JMML, neurofibromin influence
changes of ras-GTP from inactive to active (mouse
model and patient data) - higher level of ras-GTP - hypersensitive
to GM-CSF signaling
* NF1 heterozygous patients prone
to myeloid leukemia with deletion of normal allele
* NF1 double (homozygous field progenitors)
are hypersensitive to GM-CSF (transplanted
into wild-type mice, have phenotype similar to JMML)
* 30% of JMML patients have NF1
mutations, 15% have clinically neurofibromatosis
- Stem cell disorder - appears in various myeloid
lineages, including erythroid, megakaryocytic,
CD34+, CD38-, but conflicting data on B and T cells
·
Therapeutic implications
- Poor response to conventional chemotherapy
- Allogeneic transplant is treatment of choice
if donor available - high relapse rate - 40%
disease free survival rate (in unrelated and matched siblings)
- Retinoids and interferons attempted with early
studies that were promising, but not
overall very good
- Farnesyl transferase inhibitors - target once
ras pathway identified
* L-744832 (Merck) not effective
in mice - only inhibits H-ras not N or K
* L744749 (Merck) better in mice,
but showed some inhibition of normal progenitors
* (Jansen)- R115777- a FTI being
used by COG
- Inhibition of GM-CSF
* E21R, GM-CSF antagonist - effective
in mouse model - binds alpha chain and not
the beta chain and blocks actual signaling
· French case
report of end-stage JMML with good response following 2 cycles
with
E21R, but resistant after third cycle - possibly due to development
of antibody
against E21R
· Working with
both British Biotech (U.K.) and Bresogen (Australia) developers
of
E21R - developing IND, have pre-IND FDA meeting in June to open
trials in the
United States - currently in Phase II trials outside of U.S. (Dr.
Emanuel)
* Synergy with anti-TNF-alpha monoclonal
antibody
- Clinical experimental pediatric trial in COG
(Dr. Emanuel)
* Phase II window/Phase III trial
format (more detail in Innovative Therapy session)
* Experimental therapeutics tested
in 2-month window up-front
* Then patients proceed to treatment
with multi-modality regimen of retinoids, chemotherapy,
splenectomy, and stem cell transplant
* Experimental therapies include
Jansen's R 115777- FTI (only 9 patients currently
enrolled, 8 received FTI)
* In design, if response seen with
FTIs, up to 36 patients will receive on experimental
phase II agent, and then move onto next stage of protocol
* If trials opened with E21R in
the U.S., it may replace FTI in the experimental window
* Blinded study - no major toxicities
reported to date; masked to any response data
* What is dose of FTI triple seven
in infants - start at 200 move to 300 if acceptable
side effect profile is seen, special powder packet formulation
* Has the new GM-CSF conjugated
immunotoxin used in adults with AML been considered
for this trial?
· Yes, evaluated
with Dr. Frankel - does have effectiveness in vitro
· In early trials
with GM-CSF diphtheria fusion toxin in adults, there was significant
hepatotoxicity - if this issue can be overcome, it could be attempted
in children
· Currently,
this is behind development of E21R for children
* Banking being done with this protocol
as well
·
Discussion summary (Dr. Kaufmann)
- Does JMML represent a successful plan which
can be used for all the myeloproliferative
disorders?
* In an extremely rare disease,
signaling abnormalities are being fairly well characterized
* Through work in Cooperative Group,
it has been possible to do innovative trials based
on signaling
- JMML has some heterogeneity in molecular pathogenesis
* As research progresses, review
various molecules to figure out which had impact
on response
* Current estimates for mutations
from small series (Dr. Emanuel)
· 20% with ras
mutation
· 30% with NF1
· ras and NF1
are mutually exclusive
· mutation in
other 50% still to be characterized
· evaluating
which therapies have better/worse response in NF1 patients
· correlative
studies will also help answer remaining questions
- Suspect similar challenges to identify mutations
in other MPDs as in this disorder
(Dr. Gilliland)
* At signaling level, hard to identify
NF1 deficiency as consequence on ras-GTP loading
* Difficult to identify biochemical
surrogates for activation of ras map kinase pathway
in heterozygotes
* Data on ras wild-type allele -
when expressed from its own promoter, as opposed
to over-expressing it, hard to demonstrate down-stream activations
- Need to approach pathophysiology using multiple
strategies including signal transduction
and direct sequencing of as many candidates genes as might appear
to be reasonable as a rational approach to solving these problems
ESSENTIAL
THROMBOCYTOPENIA (ET) (DR. KAUFMANN)
·
Similarity to other MPDs - present with thrombocytosis, then go
on to fibrosis
·
Consider ET as distinct entity with fibrosis as opposed to
idiopathic myelofibrosis where no clear mechanisms
for pathogenesis were determined from earlier discussion
·
Evidence of abnormal pathophysiology
- Forced thrombopoietin (TPO) over-expression
in bone marrow cells - disease initially
manifests as elevated platelets and neutrophils (mouse model)
- TPO mutations
* Familial ET - Mutations in TPO
gene found in splice junction or in 5 prime untranslated
region of mRNA - extremely rare syndrome (1-2% of ET cases)
* Spontaneous ET - TPO levels reported
normal
- c-MPL mutation in TPO receptor
* Lack of constitutive activation
of c-MPL in ET - caveat reminder - very difficult to demonstrate
abnormal signaling even when thought to exist
* Research experience has not found
c-MPL mutations in TPO receptor
* c-MPL levels often report to be
normal or low, rather than elevated - not even over-expression
of a normal receptor that contributes to pathophysiology
- Megakaryocyte colony growth (CFU-Meg)
* Autonomous megakaryocyte colony
growth (CFU-Meg) - question whether CFU-Meg
can be blocked by anti-TPO reagents - is it TPO responsive or
not?
* CFU-Meg reported as hypersensitive
to IL-3 and PEG-rHu MGDF (a TPO derivative
or truncated TPO, which is pegalated megakaryocyte growth and
differentiation factor)
- Is ET a heterogeneous group of diseases where
the signaling defect has been missed?
Is ETa signaling disorder at all? (Dr. Kaufmann)
* Heterogenous disorder - many factors
turn platelet production on - no clonal marker
identified unless informative female program is used (Dr. Spivak)
* Symptomatology and clinical history
· acute patients
- from high platelet count into myelofibrosis, enlarged spleen,
acute
leukemia and death
· chronic patients
- 20 year history of high platelet counts where nothing more significant
happens (finger and toe pain, headache, bleeding)
* Define clonality in ET patients
- reduce heterogeneity of population
- Nature of clonality
* Originally ET thought to have
clonal hematopoiesis
* Research shows ET to have polyclonal
hematopoeisis in substantial fraction of patients
- disorder where clonality is in question
·
Establishing the nature of signaling abnormalities
- Lack of c-MPL constitutive activation in ET
- Hypersensitivity of CFU-Meg to IL-3 and PEG-rHu
MGDF
- Proposed Banking of Tissues (Dr. Kaufmann)
* If specimens are banked, clonality
must be established
* Also work on platelet RNA expression
which indicates that some ET patients have
clonality confined to the megakaryocyte compartment - interpretation
of available data (Dr.
Gilliland)
* If banking specimens, what is
the best tissue to try to obtain?
· Based on data
from PV, bone marrow would have both clonal and non-clonal populations
(Dr. Spivak)
· Theoretically
if disease is clonal, then peripheral blood should only reflect
the malignant
clone
· Platelets are
easy to collect
· In ET patients,
CD34 positive cell would be optimal, but hard to obtain without
IRB
approval to allow larger volume quantities for blood draw
· Might ET patients
have mutations with later progenitors than a CD34 progenitor?
Does every ET patient have a genetic lesion in their CD34 cells?
(Dr.
Gilliland)
· Since congenital
one can be seen - no reason to say no on that basis (Dr. Spivak)
* Sytematic approach for signaling
pathways (Dr. Emanuel)
· Considering
heterogeneity and difficulties with NF1 signaling, controversial
results
should not be dismissed quickly
· c-MPL activation
not yet seen - develop more systematic approach
· Examine cells
showing TPO hypersensitivity in vitro - look carefully at those
signalling
pathways - may be subsets to this disorder
· Need to conduct
study prospectively - discover TPO hypersensitivity two weeks
after receiving sample (Dr. Kaufmann)
· Also conduct
study sequentially - once first step is identified, obtain more
samples
from those informative patients and look forward
- Final Comment regarding Cooperative Group/organizing
collection of tissues
* Recommend formulating pilot treatment
protocol central to Cooperative Group - not
rigid randomization procedure, flexible design to accommodate
new treatments - providing
structure and assuring comparable results
* Obtain specimens initially as
well as throughout protocol treatment
Ability to examine new
treatments when good responses are observed or when a
particular abnormality is identified
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