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					                             BioMarker
Definition:

   Biomarker is a substance used as an indicator of a biologic state

              Existence of living organisms or biological process.
              A particular disease state
              A fragment of DNA sequence


       Proteins
       Nucleic acids

       Carbohydrate

       Lipids

       Small molecules
                             Biomarkers
Location of the biomarker:

    Location of a particular molecule can also be a marker

       Cellular-subcellular locations

       Tissue or organ locations

    Thymidylate synthase (TS)
       nucleus vs the cytoplasm
            The higher the level in NE, lower level in the cyto.
            Lower survival in colorectal cancer patients
    Galectin-3
            Galectin-3 is a member of lectin family. It is wildly distributed
            In tissue of epithelial, fibroblast, and dendritic cells.
      Blood Galectin-3
            Predict the outcomes for patients with symptoms of heart failure
                      Biomarker
Detection of biomarker

       Detection of biomarker – diagnosis
               Self properties, e.g enzymatic activities
               Antibodies, IHC, ELISA


       Detection of biomarker
               Quantitative
                 a link between quantity of the marker and disease
               Qualitative
                 a link between exist of a marker and disease
                      Biomarker & Diagnosis
Ideal Marker for diagnosis
    Should have great sensitivity, specificity, and accuracy in reflecting total
    disease burden. A tumor marker should also be prognostic of outcome and
    predictive of tumor recurrence and effectiveness of anti-cancer treatments.

Biomarker for Screening
   •The marker must be highly specific, minimize false positive and negative
   •The marker must be able to clearly reflect the early stage of disease
   •The marker must be easily detected without complicated medical
    procedures. The disease markers released to serum and urine are good
    targets for application of early screening.
   •The method for screening should be cost effective.

 Samples for biomarker detection

    Blood, urine, or other body fluids samples

    Tissue samples
Biomarker Discovery and Validation

Correlation: a biomarker vs a disease or status of a disease
        Do not need understand functions

Detection: Detection of a particular marker is important


Validation: Build statistical correlation – large number of samples

Validation: sensitivity and specificity

Validation: Stand alone vs along with other markers
                   Common Serum Markers for Cancer
                        Diagnosis/prognosis
             AFP   CEA   CA15-3   CA19-9   CA125   PSA   PSAf   PAP   hTG   HCGb   Ferr   NSE   B2M   A2M
Lung               x     x        x        x                                x      x      x
Pancreas     x     x     x                 x                                x
Kidney       x     x                                                        x                   x
Breast             x     x                 x
Ovarian      x           x        x        x                                x                   x
Cervical     x                             x
Uterine            x              x        x                                x
Prostate                                           x     x      x                               x     x
Liver              x     x        x        x                                x
Gastro             x              x        x
Colon              x              x        x             x                  x
Bladder                                                                     x
Brain                                                                       x
Leukemia                                                        x                  x            x
Myeloma                                                                                         x
Thyroid                                                               x                   x
Testicular   x                                                  x           x      x
            Common Biomarker for Cancer
AFP (>100 ng/ml)
Alpha fetoprotein (AFP) is typically found in the developing fetus.
Because of the association of the rapid cell growth, this fetal protein is
also used as a tumor marker. non cancerous liver diseases such as
cirrhosis and viral hepatitis can lead to high level AFP

CEA (>10 ng/ml)
Carcinoembryonic antigen (CEA) is produced during fetal development. After
birth, the production of CEA stops and is undetectable. CEA has also been
found elevated in nonmalignant tumors such as pleural effusions. Elevation of
CEA after conventional treatment of neoplasms has been correlated with a
recurrence of cancer

CA19-9 (>37 U/ml)
Carbohydrate Antigen 19-9 (CA 19-9) is present in the fetus in the epithelium
of the fetal stomach. It is primarily used as a marker for pancreatic cancer.
High levels also exist in conditions such as non-malignant liver disease and
other disorders of the gastrointestinal tract.
            Common Biomarker for Cancer
hTG (>10 ng/ml )
Thyroglobulin (hTG) is a glycoprotein that is found in thyroid gland. This
protein binds to thyroxine, which controls the rate of metabolic processes
in body.

HCG (>10 mIU/ml )
Human Chorionic Gonadotropin-beta (HCG) is normally produced by the
placenta during pregnancy, an indicator of pregnancy. The protein can be
detected in serum or urine. Non-malignant elevations may be observed in
pregnancy, ulcers, duke’s disease, and cirrhosis. Levels of HCG are useful in
monitoring the effectiveness of treatment.


Ferr (>120 ng/ml )
Ferritin is an iron binding storage found in the liver, spleen, and bone marrow.
Elevated levels observed in non-cancerous conditions include rheumatoid
arthritis and anemia
            Common Biomarker for Cancer

NSE (>12 ng/ml )
Neuron Specific Enolase (NSE) is produced by neurons and
neuroendocrine cells of the central and peripheral nervous system.


b2M (high level)
Beta 2-Microglobulin, (B2M), is an 11 kD protein associated with the outer
membrane of many cells including lymphocytes. It is the small subunit of the
MHC class I molecule. B2M is present in small amounts in serum, csf, and urine
of normal people. A non-malignant condition associated with high b2M levels is
pancreatitis


a2M (<500 mg/ml )
Alpha 2 Macroglobulin (A2M) is a large protease inhibitor, 720 kD, capable of
irreversibly binding and inhibiting a wide variety of proteases including
plasmin, pepsin, trypsin, chymotrypsin, and cathepsin D.
            Prostate Cancer marker PSA

PSA is a protein normally made in the
prostate gland in ductal cells that make
some of the semen. PSA helps to keep
the semen liquid. PSA, also known as
kallikrein III, seminin, semenogelase,
γ-seminoprotein and P-30 antigen, is a
glycoprotein, a serine protease


PSA has several forms in serum
        Free
        Pre-enzyme
        Complex
        Inactive
      Prostate Cancer Diagnosis with PSA
Cancer of the prostate does not cause any symptoms until it is locally advanced
or metastatic.

There is a correlation between elevated PSA and prostate cancer.

Diagnosis of PSA for prostate cancer in the most time means measure the PSA
In serum samples.

Detection of PSA allow early detection of prostate cancer.

Large screening trials have shown that PSA nearly doubles the rate of
detection possible by combine with other methods. Based on these data, PSA
testing was approved by the US FDA for the screening and early detection of
prostate cancer.

PSA is also found in the cytoplasm of benign prostate cells.

Separately measure different forms of PSA give better results
                    Prostate Marker PSMA
Prostate-specific membrane antigen (PSMA) is a type 2 integral
membrane glycoprotein. PSMA possesses glutamate carboxypeptidase
activity, Detectable levels of PSMA are also found in small intestine and brain.
Intestinal PSMA may play a role in the metabolism of dietary-glutamated
folate derivatives. Brain PSMA, also referred to N-acetylated -linked acidic
dipeptidase (NAALADase), may modulate glutamatergic neurotransmission

In addition to being abundantly and preferentially expressed on
the surface of prostate cancer cells, PSMA is also present on
endothelial cells of new blood vessels

PSMA also expressed in brush border of proximal
tubules of normal kidneys and tissue samples from
both benign and malignant renal lesions
(neovasculature)


The role of PSMA as a cancer marker is still under
investigation. PSMA is sensitive to reflect prostate cancer. The specificity, however,
is not fully verified. Most use is image the prostate metastasis by radio-isotopes
                  Prostate Marker PSMA
The role of enzymatic activity (folate hydrolase) of PSMA in
cellular transformation and metastasis is not understood.



PSMA has isoforms produced by alternative splicing.
In normal prostate epithelia, PSMA is expressed primarily as a cytoplasmic
protein termed PSM'
In prostate carcinomas, however, differential mRNA splicing leads to
expression of PSMA as a type 2 integral membrane glycoprotein possessing
a 19-aa cytoplasmic fragment, a single 24-aa membrane-spanning domain,
and a 707-aa extracellular region


Unlike PSA, PSMA is not a secret protein. Serum level is very low even
elevated. PSMA is membrane protein, ideal for targeting
   Ovarian Cancer Diagnosis by CA-125
CA-125, a product of the MUC16 gene, is a mucin made by certain cells in the
body which include those of the uterine tubes, uterus, cervix, and the lining of
the abdominal and chest cavities

CA125 is a membrane glycoprotein that has very short cytoplasmic domain
and a very long extracellular domain. The cellular function of the protein is
still unknown. The protein exist in the cells of normal and cancerous tissues
of ovarian.

The release of proteolytic fragments of CA125 leads to elevation of CA125
level in bloodstream, which is associated with progression of ovarian cancer
and a few other cancer types. The CA125 level can be over 10 folds higher
in ovarian cancer patients compared to the level in normal.

Diagnosis of ovarian cancer is often simple. A pelvic mass is suspicious and
is very often associated with ovarian cancer. However, when the pelvic mass
is detected, the ovarian cancer usually has already advanced to quit
advanced stage. There is no symptom for ovarian cancer, especially during
early stage.
                                Her-2/neu
The HER (erbB) family member are
Growth factor receptors.

In the HER (erbB) family, only Her-2
did not have identified ligand.


Expression level correlates with growth
of breast cancer cells.




Diagnostic/prognostic applications.
HER2-positive metastatic breast cancer have a more aggressive disease, greater
likelihood of recurrence, poorer prognosis and decreased survival.
             Beta-2-microglobulin (b2M)
B2M is the small subunit of the MHC class I
molecule.

How does the protein promotes tumor growth
and metastasis – the expression of MHC
class 1 in many tumor cells are repressed.

An hypothesis is that b2M is a signaling
molecule that linked to cell growth and
survival pathway.


What are the target of the signal? Receptors?
Known or unknown pathway?
            Biomarker & Targeted Imaging

Imaging probe link to a targeting moiety that target to bio-marker


Basic requirement: the marker must presented on cell surface in high number




 Ideally:
            The interaction between marker and targeting moiety is strong
            The interaction is highly specific
            The target internalization – high payload
            Distribution at neovasculature, minimal tissue penetration
             Imaging Targeting PSMA
Antibody is targeting moiety

    Antibody conjugated quantum dots



               Antibody
               Imaging Targeting PSMA

Antibody conjugated to radio-labels




                       Patient Images
                        No resolution – hardly tissue image
                        High sensitivity – good for detecting metastasis
                       MR Image Target Her-2
Advantage for both
Her-2 and PSMA
imaging is that the high
number and distribution
of the marker in
agiogenesis tissue

Disadvantage for both
Her-2 and PSMA
imaging is that the ligand
for Her-2 and PSMA are
not identified. Antibodies
are the only choice



The levels of both Her-2 and PSMA correlate well with aggressiveness of
disease
              Therapies Target Biomarkers

Simply target the biomarker by antibody

Therapeutic agent link to a targeting moiety that target to bio-marker

Basic requirement: the marker must presented on cell surface


  Ideally:
             The interaction is highly specific.
             The target internalization, accumulation good # of agent.
             Distribution at neovasculature, minimal tissue penetration.
             The level of marker correlate with disease progression
                Therapy Target Her-2

Herceptin
   Herceptin binds to HER2-positive cancer cells and may block them
   from dividing and growing.

   Herceptin attaches to the HER2-positive cancer cells and may
   signal the body's immune system to destroy the cell.


   Herceptin can also conjugated with chemotherapy (paclitaxel)
   to destroy HER2-positive cancer cells.
Herceptin




     100%   33%      10%
    Mouse   Fused   Humanized
Mechanism of Herceptin
Molecular Mechanism for Her-2 Antibody
           Therapy Target Her-2

Small Molecules target HER-2

    erlotinib (Tarceva), a kinase inhibitor for EGFR/HER1
    Target HER-2



Affibody target HER-2
     Polypeptides of 58 aa derived from IgG binding domain
     of protein A, forming a three-helix bundle.
                       Therapy Target PSMA

One important advantage is the quick
endocytosis of the PSMA upon the
antibody binding.

Due to main neovascular distribution,
targeting of PSMA is very easy, less
tissue penetration.

Both Her-2 and PSMA have a good
property is their high level correlate
well with aggressiveness of disease.
Compounds Target PSMA
    PSMA Target for Tumor Blood Vessel



PSMA is found highly expressed in neovascular of many cancer types,
especially the new vessel in cancer mass.




PSMA is an excellent target for delivery to many cancer
Cancer in Stem Cells or Progenitor Cells
Cancer Stem Cell Target
            Drugs Target Cancer Stem Cells
Company      product        Comp type   Status        Cancer           Targets

GlaxoSmithKli Tyverb        Small
                                        FDA approved Breast cancer Unknown
ne            (lapatinib)   molecule
              Omacetaxine   Small                     Chronic          Protein
ChemGene Mepesuccinat       molecule    Phase 2/3     myeloid          synthesis
              e                                       leukemia
                                                      Chronic          Telomerase
Geron (Menlo
                            Small                     lymphocytic      inhibitor
Park,        GRN163L                    Phase 1/2
                            molecule                  leukemia,
California)
                                                      solid tumors
Stemline                                              AML and          IL-3R
                       Recombinant
Therapeutics SL-401                  Phase 1          myelodysplasti
                       protein toxin
(New York)                                            c syndrome
OncoMed                                                                Unknown
                                                      Multiple solid
Pharm/       OMP-21M18 mAb           Preclinical
                                                      tumors
GSKline
                                                      Prostate and     Pan/ADAM-
Raven/Vaxgen RAV17/RAV18 mAb            Preclinical
                                                      colon cancer     9
                                                      Leukemia,        CD44
Arius
             ARH460-16-2 mAb            Preclinical   breast, colon
Research
             Imaging and Therapeutic Agent




The most significance of the double agent is the immediately follow the effectiveness
of treatment. Also follow whether the therapies have been targeted to the sites
                  Diagnosis by Proteomic
No single marker can
accurately reflecting a
disease. Not even a
panel of markers.



We need analyses a
large number of disease
markers.