E. R. Flotte
MD, 2009
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Neurosurgical
Research
·
“Biomedical
Publication for Neurosurgery Residents”, Berger MS N9/00
·
Nationwide
Inpatient Sample (NIS): www.ahcpr.gov/data/hcup/nisintro.htm
·
Clinical
Trials: N2/04.
·
“2001: Things to
come” Apuzzo MLJ N10/01
Genetics
(-) = tumor suppressor; (+) = oncogene
Cell cycle proteins
P53: chr17, G1 police
(-); LGA > GBM. Gain of function mutations lead to stable protein, prevents
apoptosis
MDM2: inhibits p53(+)
P21: p53 induces to
cause arrest(-)
P27/kip1: G1 inhibition(-)
P16/p15: inhibit CDK4/6(-)(genes CDKN2A/B)
CDK4/CDK6:
phosphorylate RB(+)
RB: phopsphorylation releases E2F allows G1>S (+)
P14ARF: alternative
reading of CDKN2A; RB independent arrest(-)
Growth Factors and Receptors
EDGFR: amplified or overexpressed in primary GBM
PDGFR
DCC: lost in
progression
PTEN(MMAC1): (-)
Migration
“Go
or Grow” hypothesis: cells postmitotic
before migration.
4 medulloblastoma
cells lines divided while migrating (N7/03).
Factor
potency: SF/HGF > TGFα > FGF1.
Scatter Factor (SF)/ Hepatocyte Growth Factor (HGF)
·
Receptor
is MET (c-Met oncogene product)
·
Potent
migratory factor
Ephrins
·
Cell-surface
bound
·
Bind
Eph receptor TKs
·
Regulate
axon guidance, neural crest migration
Matrix
metalloproteinase inhibitors: Marimastat, prinomastat
·
Gliomas produce PDGF (A & B), EGF, TGFα,
IGF-1.
·
Receptors
are tyrosine kinases
PDGF
·
PDGF-A,B,C,D
homodimers (AA, BB, CC, DD) or heterodimer
(-AB)
·
Receptors: PDGFRα (binds AA,BB,CC,AB), PDGFRß
(binds BB,DD)
·
Overexpression in LGA w/ p53 loss.
Expressed in almost all oligodendrogliomas.
·
Simian
sarcoma virus is viral homologue of c-sis/PDGFB
·
Involved
in angiogenesis
EGF
·
EGFR
in ErbB family; also binds TGFά
o
Transmembrane glycoprotein,
intracellular TyK domain
·
Neurotrophic, development,
angiogenesis
·
EGFRvIII: mutation in extracellular domain
leads to constituitive activation
·
May
be due gene amplification, overexpression, autocrine/paracrine ligand stimulation, or constitutive activation
·
EGFR
in gliomas
o 50%
GBMs overexpressed/amplified (de novo GBM). 50% of
these cases have mutations
o EGFR
mutation can occur without overexpression of wt-EGFR
o
EGFRvIII: most
common mutant in GBM. Constitutive autophosphorylation.
Oncogenic transformation in absence of ligand
·
EGFR
Antagonists: N6/04
FGF
·
Acidic
FGF (aFGF), basic FGF (bFGF),
and FGF-3 to -23
·
4
receptors, 1-4
·
Involved
in angiogenesis, neurotrophic
TGF-ß
·
Family:
BMPs, Mullerian inhibiting substance
·
Receptors:
I & II. Serine/Threonine kinases.
·
Released
by glioma cells in vivo & in vitro.
·
Inflammation,
wound healing, angiogenesis
TNFα
·
Inflammation, angiogenesis
·
Produced
by monocytes/macrophages
Receptor Tyrosine Kinases
·
600
protein kinases, 130 protein phosphatases.
Kinases are commonly in oncogenic
pathways
·
Frequent
Alterations in Cancer:
–
Genomic
rearrangements (e.g. Bcr-Abl, EGFR)
–
Mutation
(e.g. Flt-3, c-Kit, c-Met)
–
Overexpression (e.g. EGFR, VEGFR, c-Met)
·
Composed
of: Extracellular ligand-binding domain, membranous
domain, intracellular domain with intrinsic tyrosine kinase
activity
·
Ras > Raf > Mek > MAPK
o
Most
RTKs. Needs assistance of scaffolding proteins.
o
Ras inactiviating gene
neurofibromin mutated in NF1;
·
PI-3
Kinase (PIP2 > PIP3) > PDK1/2 > Akt
o
Used
by EGFR
o
PTEN
inhibits PI3K
o
Akt inhibits apoptosis, stimulates cell growth
& proliferation
·
PLC-γ
(PIP2 > IP3 & DAG)
o
Used
by EGFR
·
STATs
o
EGFR
activates STAT1,3, & 5
o
STAT1
proapoptotic, antimitotic;
STAT3 antiapoptotic, promitotic
·
Mitogen-activated protein kinase
·
Cause
DNA synthesis and cell division
·
RTK
inhibitors:
o
Herceptin, HER-2 antibody (to
extracellular domain). Approved for breast cancer – 15% response rate.
Hesselager N903
·
Inhibitors
of tyrosine kinase activity: Iressa,
Tarceva – EGFR; Gleevec –
PDGFR (in clinical trials for GBM)
·
INK4A-ARF
locus codes for p16 & p14 suppressors. Deleted/ inactivated in 60% of GBM.
·
p53:
on Chr17
·
Rb: inhibited by SV40 T antigen T121.
·
MYC:
transcription factor leading to cell proliferation, Amplified in some medulloblastomas.
Ubiquitin-Proteosome
Pathway
Proteosome inhibitor PS-341
Apoptosis
Telomerase
Ribonucleoprotein enzyme
that replicates telomeric DNA. Reactivated
in some cancers, including GBM.
hTERT: human telomerase
reverse transcriptase. May predict prognosis.
Sonic Hedgehog
Receptor:
Patched (PTCH)
Gli – downstream effectors
Mutated
in minority of gliomas
· Reviews: ”Angiogenesis in Nervous System Disorders” Zadeh G N12/03, “Angiogenesis and Gliomas” Jouanneau E N1/08
·
Vasculogenesis: new blood vessel
formation (embryonic).
Angiogenesis: New
branches formed off existing vessels (tumors, etc)
·
Glioma angiogenesis: VEGF (primary), SF/HGF,
bFGF most important
·
Edema:
VEGF & angiopoietins
VEGF
·
endothelium specific angiogenic factors.
·
5
homologues (B,C,D,E, and placenta growth factor PlGF).
·
Receptors:
VEGFR-1, VEGFR-2, neuropilin-1 (Nrp-1)
·
Promotes
NO synthesis, vascular permeability, proteases. May be responsible for edema.
·
VEGF
in gliomas:
o
VEGF is
expressed in human high-grade glioma but not in
normal brain
o
VEGF receptors are expressed only on
endothelial cells
o
Increased
expression of VEGF, and VEGF receptors correlates with tumor vascularity and malignancy grade in human gliomas
o
Upregulation of VEGF is associated with areas of necrosis
o
High proliferation
rate of malignant glioma may lead to areas of
necrosis and hypoxia and result in increased expression of VEGF
Angiopoietins
·
Ang-1
to 4. Ligands for Tie-2, endothelium specific
tyrosine kinase receptor.
Endostatin/angiostatin:
Inhibit angiogenesis
Also
thrombospondin 1 & 2, platelet factor-4
Therapy: VEGF antisense,
Thalidomide: inhibits endothelial cell proliferation, Fumagillin,
angiostatin, endostatin,
COX2 inhibitors (Celecoxib), Topotecan
(VEGF antagonist)
Angiogenesis
N12/03
Tumor Associated
Antigens
·
Gliomas have been shown to express several tumor
associated antigens (TAAs): EGFRIII, glycoprotein 240, tenascin,
survivin, squamous cell
carcinoma–associated reactive antigen for cytotoxic T
cells 1, α-2 chain of interleukin-13 receptor, and melanoma-associated
antigens, such as tyrosinase, tyrosinase-related
protein 1 and 2, glycoprotein 100, melanoma antigen-1, and melanoma antigen-3.
·
Identification
of a universally expressed glioma TAA has not been
identified. Tumor cell clones that do not express the particular, targeted TAA
(anymore) escape from the immune rejection and, thus, have an important
proliferation advantage.
Types of
Immunotherapy:
Cellular
·
Antigen
Presenting Cells (APCs) present antigens to T-cells via MHC
·
MHC
not detected in CNS
Cytokines
·
TNFά,
IL-2, IL-4, INF-άßγ.
·
No
clear survival improvement when administered to GBM patients. IL-2 & INFά
showed significant
toxicity.
Adoptive
·
Intratumoral injection of
peripherally harvested lymphocytes or TILs (tumor infiltrating lymphocytes),
stimulated ex vivo with IL-2
·
No
definite survival benefit
·
Human,
non-MHC restricted, cytotoxic T-cell leukemic cell
lines (TALL-104) – cytotoxic against tumors, spares
normal tissue
·
Stimulation
of autologous lymphocytes with patient’s tumor cells
Passive
·
Intratumoral administration of
antibodies leading to cytotoxicity, or conjugated to
toxin or radioisotope
- Antigens: tenascin, EGFR, EGFRvIII
(Mab806), IL-4, transferrin receptor (no glioma specific antigens identified yet)
- Toxins: Pseudomonas,
Diptheria
- Radioisotopes: I131
·
Ex:
I131 conjugated anti-tenascin, in Phase II
trials
·
IL-4
conjugated to pseudomonas exotoxin, delivered by
convection-enhanced delivery in Phase I trials.
·
IL-13
also used.
The types
of vaccines listed below represent various methods investigators have devised
for presenting cancer antigens to the body's immune system. This list is not
meant to be comprehensive.
Antigen/adjuvant
vaccines
Antigen vaccines were some of the first cancer vaccines investigated. Antigen vaccines
commonly use specific protein fragments, or peptides, to stimulate the immune
system to fight tumor cells. One or more cancer cell antigens are combined with
a substance that causes an immune response, known as an adjuvant. A cancer
patient is vaccinated with this mixture. It is expected that the immune system,
in responding to the antigen-carrying adjuvant, will also respond to tumor
cells that express that antigen.
Whole
cell tumor vaccines
Taken either from the patient's own tumor (autologous)
or tumor cells from one or more other patients (allogeneic),
these whole cell vaccine preparations contain cancer antigens that are used to
stimulate an immune response.
Dendritic cell (DC) vaccines
Specialized white blood cells, known as dendritic cells (DCs), are taken from a patient's blood
through a process called leukapheresis. In the
laboratory, the DCs are stimulated with the patient's own cancer antigens,
grown in petri dishes, and re-injected into the
patient. Once injected, DC vaccines activate the immune system's T cells.
Activation by DCs is expected to cause T cells to multiply and attack tumor
cells that express that antigen.
·
DCVax®-Brain (
o
Clinical
trial data to date in brain cancer patients have shown that DCVax(R)-Brain
delays disease recurrence by nearly 3-fold, from 6.9 months to 18.1 months for
newly diagnosed patients. DCVax(R)-Brain also extends
these patients' survival from 14.6 months to more than 33 months (and
continuing -- median not yet reached). In parallel with making DCVax(R)-Brain commercially available to patients at
selected medical centers in
Viral
vectors and DNA vaccines
Viral vectors and DNA vaccines use the nucleic acid
sequence of the tumor antigen to produce the cancer antigen proteins. The DNA
containing the gene for a specific cancer antigen is manipulated in the
laboratory so that it will be taken up and processed by immune cells called
antigen-presenting cells (APCs). The APC cells then display part of the antigen
together with another molecule on the cell surface. The hope is that when these
antigen-expressing APC cells are injected into a person, the immune system will
respond by attacking not only the APC cells, but also tumor cells containing
the same antigen. Vector-based and DNA vaccines are attractive because they are
easier to manufacture than some other vaccines.
Idiotype vaccines
Because antibodies contain proteins and carbohydrates,
they can themselves act as antigens and induce an antibody response. Antibodies
produced by certain cancer cells (i.e., B-cell lymphomas and myelomas), called idiotype antibodies, are unique to each patient and can be
used to trigger an immune response in a manner similar to antigen vaccines.
·
Dendritic Cell Vaccination: See deVleeschouwer N11/06
·
As
opposed to the other antigen-presenting cells, DCs are able to present antigens
in both MHC Class II and I molecules. In this way, they can prime both CD4- T
helper cells and CD8- cytotoxic T-cells
·
The glycosphingolipid GD2, a disialoganglioside
normally on the cell membranes of neurons, is expressed on the surfaces of a
wide range of tumor cells including neuroblastoma, medulloblastomas, astrocytomas, melanomas,
small-cell lung cancer, osteosarcomas and other soft
tissue sarcomas
·
Glioma cells in culture lose GFAP, acquire fibronectin, become fibroblast-like (spindle-shaped)
·
9
of 68 GBM and 1 of 19 Grade 1-2 astrocytomas GFAP (+)
·
Formalin-fixed,
parafin-embedded sections suboptimal for staining
most tumor antigens
·
Better
to use fresh cells through flourescence-activated cell sorter (or staining freshly
frozen samples) (Linskey 1997)
·
Animal
serum does not reflect true environment, better to use astro-CM
Transplantation
·
Cultured
human (xenograft) or rodent (allograft) tumor cells
implanted into immunodeficient mice
·
Reproducible,
but not infiltrative, cultured cells genetically altered
Chemical mutagenesis
Transgenic Mice
·
Gain-of-function
(transgenic mice) or loss-of-function (targeted deletions)
·
Germ-line mutations: Requires cross-breeding. Expensive
·
Conditional models: Somatic cell gene transfer with retroviral
vectors
o
MMLV: replication competent, infects proliferating cells
o
Tv-a-RCAS:
§
RCAS:
avian-leukosis virus based vector, contains oncogene.
ALV is a replication deficient retrovirus
§
tv-a: The RCAS receptor,
which is inserted after a specific promoter (eg nestin, GFAP) in transgenic mice.
§
Allows
for tissue or cell-specific transfection. The RCAS
vector is injected into transgenic mouse and transfects
cells targeted by the promoter (i.e. GFAP+) and leads to protein production in
only those cells.
§
Can
only produce proteins, not delete them (but can produce inhibitors – i.e. SV40
T121 & Rb).
§
N-tv-a mice crossbred with Ef-luc
mice to create fluorescent mouse tumor models (N1/05)
·
Knockout
rats (N8/03)
Signal
Transduction Pathways
·
Transduction
of PDGF-B by a replication-competent
vector (MMLV) causes formation of variety of tumor types, including GBM
·
Transduction
of PDGF-B by replication-incompetent vector (ALV) into nestin
(+) CNS progenitors causes formation of oligodendrogliomas. Transduction into astrocytes
forms mixed gliomas.
·
Transgenic
mice expressing v-erbB (EFRR homologue) from S-100B
promoter (astrocytes & glial
precursors) form oligodendrogliomas.
·
In
RCAS-TVA system, transfer of EGFR does not form gliomas
unless combined with cell cycle disruption.
·
Overexpression of RAS from GFAP
promoter induces GBM
·
Overexpression of v-Src from GFAP promoter induces astrocytomas
·
Germline RAS or v-Src
overexpression has normal development, develop gliomas by secondary mutation
·
Combined
activation of K-RAS and AKT pathways sufficient for GBM formation in nestin (+) progenitors
·
Either
alone is insufficient
·
RAS+AKT
in differentiated astrocytes does not form gliomas
·
Mutation
in cell cycle genes did not increase gliomagenesis
Cell
cycle disruption
·
INK4A-ARF
-/- mice do not develop gliomas
·
Requires
RAS+AKT mutation
·
p53
-/- mice rarely give rise to gliomas (most die of
lymphomas or sarcomas early)
·
p53+Nf1
deletion induces GBM
·
NF1
inactivates RAS
·
Deletion
of p53 or Rb in GFAP (+) cells do not develop gliomas
·
Disruption
of either cell cycle pathway itself weak oncogenic
factor, insufficient for gliomagenesis
·
Mutations
in cell cycle pathways do appear to be sufficient for tumor progression
·
Increased
malignancy and shortened survival seen in Ink4a-Arf -/- mice compared to normal
in S100B-v-erbB and PDGF models
Cooperation
between cell cycle and signal transduction pathways
·
RAS+AKT
in INK4A-ARF -/- mice forms gliomas
·
p53
+ NF1 mutations
Convection Enhanced Delivery (CED, Clysis)
·
Therapeutic
agents are delivered through catheters driven by pumps. The agents diffuse
through the surrounding brain.
·
Multiple
agents, icluding liposomes,
viral vectors, radioactive ligands, and toxic ligands are under investigation for use with CED.
·
Cintredekin besudotox (IL13-PE38QQR)
(NeoPharm Inc.) is IL-13 fused to Pseudomonas exotoxin
which binds selectively to interleukin-13R2)
receptors overexpressed by malignant gliomas. Delivered via CED. Vogelbaum MA N11/07
·
Two
randomized trials (Precise and TransMID) failed to
show a benefit of CED over standard therapy.
Bioreactors
·
20-800μm
·
Capsule:
Alginate
·
Cells:
primary postmitotic, immortalized pheochromocytoma
P-12, fibroblasts (baby hamster kidney)
·
Immunoprivledged
·
Used
to deliver endostatin in rat & mouse glioma models (Joki 2001);
disappointing Phase I results
Tumor Staining
·
Fluorescein Sodium – IV, with or
without microscope fluorescent filter
·
5-aminolevulinic
acid – metabolized to protoporphyrin IX by tumor
enzymes – requires fluorescent filter
BBB Disruption
·
Mannitol, RMP-7
·
Blood-Brain
Barrier: N1/04.
Bioinformatics
·
The National Cancer Institute's Molecular Targets
Development Program (MTDP) to identify and evaluate molecular targets that
may be candidates for drug development
Miscellaneous
·
Annexin VII: Chr 10q21. Stronger
predictor of GBM survival than any other variable (JN11/03)
·
Proteomics: see Jagannathan J N1/09
Gene
Therapy Primer Chiocca EA N8/03
Advanced
Cancer Genetics N11/03
Induction
Neurogenesis: N1/04.
Cell
cycle: N3/04
RTK
N5/04 (N9)
Gene
Therapy:
mAbs, immunotoxin
conjugates, antisense oligonucleotides, ribozymes
12
genes amplified in GBM: EGFR, cyclin-dependent kinase 4, murine double minute 2,
sarcoma-associated sequence.
Molecular
Genetic Imaging
·
Brahma-related
gene-1 (Brg-1) negative mutants resulted in the development of embryos with
smaller brains containing neurons but virtually no glial
cells. When they isolated neural stem cells, placed them into cell culture and
then removed Brg1, the cells in the culture turned into neurons but failed to
differentiate into glia. (Larry Sherman Developmental
Biology)
Meningiomas
See
Neurologist 10:3 5/04.
Medulloblastoma
·
Developmentally
Regulated Genes in Medulloblastoma (Yokoto 04): Unc33 Like Protein (ULIP), SOX4, OTX2, Neurnatin
·
Medulloblastomas mutated at 17p
distal to TP53 – unknown gene.
Neuroregeneration and Stem Cells
·
Glial Derived Neurotrophic
Factor (GDNF) infused by clysis into putamen in Parkinsons pts 39%
improvement in UPDRS (N8/03)
Reviews:
·
Barami K N9/00.
·
Tator C N11/06.
·
Farin A N1/09 – Foundations and Landmarks in Stem Cell
Biology
Human Marrow Stromal Cells (hMSCs).
·
Secrete
growth factors. Used in TBI, CVA.
Human SVZ
·
Thin
ribbon multipotiential astrocytes,
no RMS, no migratory neuroblasts (Sanai,
Nature 427:740, N4/04)
Stem Cell plasticity: N8/03
\
Cancer Stem Cell
Pathways
·
Bmi-1: The Polycomb group
transcriptional repressor Bmi-1 was discovered as a common oncogene activated
in lymphomaand later shown to specifically regulate
HSCs. The role of Bmi-1 has also been illustrated in neural stem cells. The
pathway appears to be active in cancer stem cells of pediatric brain tumors
·
Notch: The Notch pathway has been known to developmental
biologists for decades. Its role in control of stem cell proliferation has now
been demonstrated for several cell types including haematopoietic,
neural and mammary stem cells. Components of the Notch pathway have been proposed
to act as oncogenes in mammary and other tumors.
·
Sonic hedgehog and Wnt: These developmental pathways are also
strongly implicated as stem cell regulators. Both Sonic hedgehog(SHH)
and Wnt pathways are commonly hyperactivated
in tumors and are required to sustain tumor growth. However, the Gli transcription factors that are regulated by SHH take
their name from gliomas, where they are commonly
expressed at high levels. A degree of crosstalk exists between the two pathways
and their activation commonly goes hand-in-hand. This is a trend rather than a
rule. For instance, in colon cancer hedgehog signalling
appears to antagonise Wnt
Other Spinal Cord Injury Research
·
Biodegradable
Polymer Grafts – act as conduit and for release of therapeutic agents. Friedman JA N9/02
·
Oscillating Field
Stimulation Shapiro S JNS1/05
Parkinson’s Disease Transplantation Research
·
Carotid
Body Cell Aggregate Transplantation (Arjone V N8/03)
·
Neuroprosthetics
·
Also
known as brain-machine interface, brain-computer interface, or neurorobotics
·
Motor neuroprosthetics
are machines that recognize electrophysiological alterations in the brain
representing motor intention and translate these neural signals into extant
activity to control an external device.
·
The most successful neuroprosthetic to date has been the cochlear implant, used
now by over 100,000 patients.
·
Receiving
devices, in order of spatial and temporal resolution include EEG, electrocorticography, or single-unit electrodes.
·
The
Braingate 100-electrode array (below) has been
implanted in a human patient (Hochberg Nature 2006, N10/06). Manufactured by Cyberkinetics
·
Review:
Leuthardt EC N7/06
·
Micromachines and Microelectromechanical Systems: see Roy S N10/01
Nanotechnology
·
“Neurosurgery
in the realm of 10-9” Elder JB N1/08
Vasospasm
·
Endothelin receptor antagonists: see Chow M N12/02
Psychosurgery
·
History:
Heller N10/06
·
Aminosteroids: There is no evidence
to support the routine use of aminosteroids in the
management of traumatic head injury. On the basis of the existing evidence from
randomized trials of aminosteroids in head injury, it
is not possible to refute the possibility of moderate but potentially
clinically important benefits or harms. A further randomized controlled trial
of tirilazad mesylate with
1156 participants has been completed, the results of which should become
available in the near future. (Cochrane Database)
·
Calcium Channel Blockers: The authors found
six eligible trials involving 1862 patients. The results indicate that there is
insufficient evidence to support the use of calcium channel blockers. (Cochrane Database)
·
Excitatory Amino Acid Inhibitors: The case for efficacy
of excitatory amino acid inhibitor therapy remains unproven. To date, no
product has proven to be efficacious for improving the outcomes of
brain-injured patients. Early termination, unpublished, and underpowered
studies limit a clear appreciation of the merits of this form of intervention.
Additional studies, some of which remain in progress, may more clearly define
the efficacy and effectiveness issues. (Cochrane Database)
·
Hyperbaric Oxygen: In people with traumatic brain injury, the
addition of HBOT significantly reduced the risk of death. Pooled data from the
three trials with 327 patients that reported mortality, showed a significant
reduction in the risk of dying when HBOT was added to the treatment regimen. However, there is
little evidence that more survivors have a good outcome. There was a trend
towards, but no significant increase in, the chance of a favourable
outcome when defined as full recovery,
·
Hypothermia: There is no evidence that hypothermia is
beneficial in the treatment of head injury. The earlier, encouraging, trial
results have not been repeated in larger trials. The reasons for this are
unclear. Hypothermia increases the risk of pneumonia and has other potentially
harmful side-effects. Therefore, it would seem inappropriate to use this
intervention outside of controlled trials (Cochrane Database)
·
Magnesium: There is currently no evidence to support the
use of magnesium salts in patients with acute traumatic brain injury. (Cochrane Database)
·
Monoaminergic Agonists: The authors found
three trials but none of these looked exclusively at patients with a severe
brain injury, therefore there were no satisfactory studies of the effectiveness
of monoaminergic agonists for severe TBI.
Consequently, there is, at present, insufficient evidence to support the
routine use of
Sequelae:
·
Coma: About half of people in a coma because of traumatic
brain injury will wake within a year of the accident. Sensory stimulation
methods vary greatly, from one or two hourly sessions of a day, through to
shorter sessions every hour for 12 to 14 hours a day. The review found there is
no strong evidence to determine whether sensory stimulation benefits people in
comas. (Cochrane Database)
·
Reviews: Marshall
LF N9/00
Revised 6/1/09
Text Copyright 2009