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					              Workshop #1

      Lung Cancer 101:
   Understanding the Disease

    Dr. Paul Bertics, University of Wisconsin, Madison
Section contents:
    •Speaker biography
    •Speaker presentation slides
    •Lung cancer fact sheets
Paul Bertics, Ph.D., University of Wisconsin-Madison
Department of Biomolecular Chemistry
571A Medical Sciences Center, 1215 Linden Dr.
Madison, WI 53706
(608) 262-8667
Paul J. Bertics joined the University of Wisconsin, Madison faculty in 1986 and is currently the
Kellett Professor of Biomolecular Chemistry and a member of the Executive Committee (Leader
of the Cancer Cell Biology Program) of the UW Comprehensive Cancer Center (UWCCC) and
Co-Director in the Material Sciences Research Center (MRSEC, School of Engineering).
Dr. Bertics’ research is focused on hormone action in growth control and immune function, and
he has received the Inbusch Award for Meritorious Research, the Eli Lilly Biochemistry Award,
and the Kellett Award. His publication record consists of over 110 publications and 4 patents in
the areas of cell signaling, endocrinology and biochemistry. His current funding includes five NIH
grants and one NSF grant. Dr. Bertics is or has been a member of various editorial boards (e.g.,
Endocrinology and Methods in Molecular Biology) and major grant review panels (NIH Study
Sections, an American Cancer Society Panel, and VA Merit Review Boards). Dr. Bertics has
served on international grant review panels including the Arthritis Research Campaign (UK), the
US-Israel Binational Science Foundation, the Wellcome Trust (UK), the Medical Research
Council (UK), and the Alberta Herit McCafferty Grant Program (Canada).
Dr. Bertics teaches extensively at the undergraduate, graduate and medical school levels. He
has received several teaching awards including the UW Distinguished Teaching Award-
Chancellor's Teaching Award, the UW Medical School (student-selected) Teaching Award, the
UW Medical School Distinguished Teaching Award (an alumni award), the UW Medical School
Dean's Teaching Award, the UW Medical Student Association Pacemaker Award For Teaching
Excellence (2 time recipient), and Hilldale Undergraduate/ Faculty Research Fellowships (over
10 times).

1)   General Cell Biology

2)   What Is Cancer?

3)   Lungs and Lung Cancer Biology

4)   Epidermal Growth Factor and Cancer

5)   Developing Lung Cancer Diagnostics Using Liquid
     Crystal-based Technologies
                           The Human Body
The human body is wonderfully complex and is organized into many systems.

A system is a group of organs/tissues and other structures that cooperate to
achieve a given function; many functions involve interactions between systems.

   Skeletal      Nervous        Respiratory      Endocrine         Lymph/Immune

 Digestive       Urinary       Reproductive       Muscular             Circulatory

   Organs and Tissues Are Composed of Cells
The organs and tissues of the human body are composed of cells.

Cells are the structural and functional units of all living organisms. They
consist of genetic material surrounded by cytoplasm that is enclosed by a
cell or plasma membrane.

Some organisms, such as bacteria, are unicellular, whereas other organisms,
such as humans, are multicellular (containing over 100,000,000,000,000 cells
with greater than 200 differing specializations!).

There are two general categories of cells: eukaryotes and prokaryotes.

      Plasma Membrane

          General Classes of Cells- Prokaryotes
Prokaryotes: The simplest of cells- lack a nuclear membrane. Bacteria are
the most studied prokaryotes, but a 2nd group of prokaryotes, called
archaea, has provided evidence of a third cellular domain of life.

Prokaryotes are unicellular organisms that do not develop or differentiate
into multicellular forms.

Each cell is capable of independent existence and there is little continuity or
communication between the cells.

      Plasma Membrane

           General Classes of Cells- Eukaryotes
Eukaryotes: Include fungi, animals, plants and some unicellular organisms. They
are ! 10X larger than prokaryotes & up to 1000X greater in volume.

Eukaryotes differ from prokaryotes in that they contain membrane-bound
compartments wherein specific metabolic activities occur, including a nucleus (a
membrane-defined compartment containing the cell’s DNA).

The nucleus gives the eukaryote (“true nucleus”) its name.

Eukaryotes contain other specialized structures (organelles) that perform
dedicated functions. Many different organelles are found in eukaryotes, but
we will focus on only a few with respect to their role at a molecular level.

       Plasma Membrane

              Eukaryotic Cell Structures
Plasma (Cell) Membrane- serves to separate and protect a cell from its
surrounding environment- made mostly from a double layer of proteins and
lipids. Embedded in this membrane are various sensors, channels and pumps.

Cytoskeleton (such as Microtubules)- acts to organize and maintain cell
shape; anchors organelles; helps in the uptake of external materials
(endocytosis); moves parts of the cell during growth, adhesion and motility.

Cytoplasm (Cytosol)- a large fluid-filled space wherein specific metabolic
activities occur and where the cell's organelles reside.

                     Eukaryotic Cell Genetic Material
    Genetic Material- Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most
    organisms use DNA as their genetic material, but a few viruses use RNA.

    Eukaryotic genetic material is divided into discrete units called genes.

    Human genetic material exists in 2 locations: the nuclear genome (as protein-
    nucleic acid complexes known as chromatin) and the mitochondrial genome. The
    nuclear genome is linear DNA and is divided into 23 chromosome pairs.

 The mitochondrial genome
 is a circular DNA molecule
 separate from the nuclear
 DNA. It is very small but
 codes for several key
                  Selected Eukaryotic Organelles
Nucleus- consists of a nuclear membrane around a fluid nucleoplasm containing the
DNA/chromatin. The nucleolus is a dense region of RNA and is the site of ribosome
formation. The nucleus determines the basic structure and function of a cell.

Ribosomes—complex of RNAs/proteins (cytoplasm & endoplasmic reticulum); responsible
for processing the genetic instructions carried by an mRNA. Conversion of an mRNA’s
genetic code into the exact sequence of amino acids of a protein is called translation.

Mitochondria- self-replicating organelles that contain their own genome and play a key
role in metabolizing nutrients to generate energy, often as adenosine triphosphate-ATP.

Endoplasmic Reticulum, Golgi Apparatus- transport network for molecules targeted for
certain modifications and specific destinations.

Lysosomes- enriched for
digestive enzymes, and can
contain over 30 enzymes for
degrading proteins, nucleic
acids, and certain sugars called
     Cell Division aka Growth, Proliferation or
                Cell Cycle Progression
Multicellular organisms (us!) grow and/or replace damaged or worn-out cells
through a replication process known as mitosis, which is the division of a
eukaryotic cell nucleus to produce two identical daughter nuclei.

Multicellular organisms create new cells by first replicating the cellular
constituents and then splitting into two cells, e.g., we produce new skin cells
and liver cells by replicating the DNA found in that cell through mitosis.

During mitosis, the parent cell duplicates its chromosomes, providing the
daughter cells with a complete copy of genetic information. The chromosomes
then align at the equatorial plate and the sister chromatids separate.

       The Cell Cycle


Preparation for                   G0
    Mitosis                               Resting

       G2                          G1
                                   Preparation for
                                   DNA Synthesis


                  DNA Synthesis
           Regulation of the Cell Cycle

Induction/regulation of proliferation

Programmed cell death (Apoptosis)


  " the cycle should only continue if specific parameters are met
   including: specifically timed protein expression/modification,
        proper DNA repair, complete DNA replication

Cancer = Altered or Inappropriate Cell Cycle Regulation
                                 What Is Cancer?

Cancer is the general name for a group of more than 100 diseases in
which the cells in a part of the body begin to grow out of control.

Although there are many kinds of cancer, they usually start because
abnormal cells lose proper control of their growth (cell division) and/or

Much of the following sections are excerpted from the American Cancer Society site:
    Transformation of Normal Cells into Cancer
Normal body cells grow, divide, and die in an orderly fashion.

Early in life, normal cells divide more quickly until the person becomes an adult.
After that, cells in most parts of the body divide only to replace worn-out or
dying cells and to repair injuries.

Cancer often develops because of damage to a cell’s genetic material (DNA).

Generally, when DNA is damaged (mutated), the cell either dies or is able to
repair the DNA. In cancer cells, the damaged DNA is not properly repaired.

People can inherit damaged DNA or DNA can become damaged by exposure to
environmental agents- chemicals, viruses, tobacco smoke or too much sunlight.
                  General Considerations:
              How Cancer Spreads (Metastasis)

Cancer cells have altered cell cycle
behavior, and instead of dying, they
outlive normal cells and continue to
grow and make new abnormal cells.

Cancer often forms as a tumor (a lump
or mass). Some cancers (leukemia) do
not form tumors but involve the
blood/blood-forming organs & circulate
to other tissues where they grow.

Cancer cells often travel via the blood
or the lymph system to other parts of
the body where they grow and replace
normal tissue. This spreading process is
called metastasis.

                  General Considerations:
                Nomenclature and Metastasis

Even when cancer has spread to
a different part of the body it is
still named for the place in the
body where it started.

For example, breast cancer that
has spread to the liver is
metastatic breast cancer, not
liver cancer.

Not all tumors are cancerous.
Benign (non-cancerous) tumors do
not spread (metastasize) to
other parts of the body and are
very rarely life-threatening.

              General Considerations:
        How Cancers Differ from Each Other
Cancers can begin in many parts of the body.

However, different types of cancer can act very distinctly, e.g., lung cancer
and breast cancer are very different diseases. They involve diferent cell
types, grow at different rates and respond to different treatments.

Accordingly, no single approach is as yet feasible to treat all cancers and
considerable effort is underway to target the specific defects that account
for a given individual’s cancer (personalized medicine).


     “Personalized medicine: Tailoring treatment to
     your genetic profile”
     “Gene variations are common but can have a big impact on how
     your body processes medications. See what genetic tests are
     available to help customize your treatment.”
                    General Considerations:
                         Risk Factors
A risk factor is anything that increases a person's chance of getting a
disease. Some risk factors can be changed, and others cannot.

Risk factors for cancer include age, sex, and family medical history. Others
are linked to cancer-causing environmental factors or are related to lifestyle
choices, such as tobacco and alcohol use, diet, and sun exposure.

Having a risk factor means a person is more
likely to develop cancer at some point in their
life, but having one or more risk factors does
not mean that a person will get cancer.

Some people with one or more risk factors
never develop cancer, whereas others who do
get cancer have no apparent risk factors.

 General Considerations: Selected Risk Factors

Cancers of the lung, mouth, larynx, bladder, kidney, cervix esophagus,
and pancreas can be related to tobacco use.

Skin cancer is related to unprotected exposure to strong sunlight.

Breast cancer risk factors have been proposed to include age; changes
in hormone levels throughout life, obesity, lack of physical activity,
alcohol consumption, and inherited genetic defects (BRCA1/2).

Prostate cancer risk factors include age, race, diet & genetic defects.

Environmental factors such as tobacco use, diet, infectious diseases,
chemicals and radiation cause ! 75% of all cancer cases in the US.

Certain cancers have been linked to viral infections (HPV and cervical
   General Considerations: Cancer Incidence

Over one million people develop cancer each year.

In the US, about one 1 of every 2 men and 1 out of every 3 women
will develop cancer at some point during their lifetime.

Cancer can occur at any age; however,
about 77% of all cancers are diagnosed in
people age of 55 and older.

Although cancer occurs in Americans of all
racial and ethnic groups, the rate of cancer
occurrence (the incidence rate) varies from
group to group.

      Metastatic carcinoma of the lung.
               Cancer- Signs and Symptoms
Cancer is a group of diseases that can cause differing signs and symptoms,
depending on its site, size, and how much it affects nearby organs/structures.
If a cancer spreads, then symptoms may arise in different parts of the body.

As a cancer grows, it pushes on nearby organs, blood vessels, and nerves,
creating some of the signs and symptoms. Some cancers start in places where it
does not cause any symptoms until the cancer has grown quite large.

Symptoms may include fever, fatigue, or weight loss
(cancer cells use much of the body’s energy & may
release agents that alter metabolism). Cancer may
also affect the immune system to cause symptoms.

In certain cases, cancer cells can release substances
into the blood that cause symptoms not usually linked
to a given cancer, e.g., some lung cancers release
hormones that affect blood calcium levels, affecting
nerves & muscles and causing weakness and dizziness.

                    General Considerations:
                    Staging and Treatment
Staging is the process of assessing how far the cancer has spread. Staging the
is a key step in determining treatment options.

There is more than one system for staging. The TNM system is the one used
most often. It gives three key pieces of information:

* T describes the size of the tumor, and whether the cancer has spread to
        nearby tissues and organs.
* N describes how far the cancer has spread to nearby lymph nodes.
* M shows whether the cancer has spread (metastasized) to other organs.

Letters & numbers after the T, N, M give more detail about these factors, e.g.,
a tumor classified as T1, N0, M0 is small and has not spread to the lymph nodes
or distant organs. The lower the number, the less the cancer has spread.

How is Cancer Treated? Four major types of cancer treatment are surgery,
radiation, chemotherapy, and biologic therapies.
                        What Causes Cancer?

“Several lines of evidence indicate that tumorigenesis in humans is
    a multistep process and that these steps reflect genetic
 alterations that drive the progressive transformation of normal
          human cells into highly malignant derivatives.”

Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
                    Primary Cancer Traits

Self-sufficiency in growth signals

Insensitivity to anti-growth signals

Evading apoptosis

Limitless replicative potential

Sustained angiogenesis

Tissue invasion and metastasis

                         Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
      The Process of Tumor Establishment-I
Cellular modification:
   Multiple mutations; alterations in systems that control cell growth
   (proto-oncogenes " oncogenes), tumor suppressor genes (Rb, p53, p73
   APC), DNA repair (BRCA1/2)

Altered gene expression or activity leading to cancer cellular traits

                        Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
      The Process of Tumor Establishment-II
Formation of a cellular mass:
       Increase in cell number from enhanced proliferation and/or
       anti-apoptotic behavior

Increased vascularization; tumor support, cell metastasis

                        Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
                      Lungs and Lung Cancer
To understand lung cancer, let’s first discuss normal lung structure & function.

Your lungs are two sponge-like organs located in your chest. The right lung is
divided into 3 sections, called lobes. The left lung has 2 lobes; it is smaller
because your heart takes up more room on that side of the body.

When you breathe, air enters the lungs through the trachea (windpipe). The
trachea divides into tubes (bronchi), which divide into smaller branches
(bronchioles). At the end of the bronchioles are tiny air sacs (alveoli).

Blood vessels run through the alveoli- O2 is
absorbed from the inhaled air into your blood
and CO2 from the body is released into the
alveoli & expelled when you exhale.

A lining, called the pleura, surrounds the
lungs. The pleura protects your lungs and
helps them slide back and forth as they
expand and contract during breathing.
            Start and Spread of Lung Cancer
Most lung cancers start in the bronchi, but can also begin in the trachea,
bronchioles, or alveoli. They may develop over many years and may start as
areas of pre-cancerous changes in the lung. These changes happen within the
cells, but do not yet form a mass or tumor.

The pre-cancerous changes may progress to
true cancer. As they develop, the cancer
cells can make chemicals that cause new
blood vessels to form (angiogenesis).

These new blood vessels nourish the cancer
cells, which can continue to grow and form a
tumor large enough to be seen on imaging
tests such as X-rays.

At some point, cancer cells may be released
from the original tumor & spread to other
parts of the body (metastasis).

       Lung Cancer and the Lymphatic System
The lymphatic system is one of the ways in which lung cancers can spread.

Lymph nodes are small, bean-shaped collections of immune system cells that
are connected by lymphatic vessels, which carry a clear fluid called lymph
(instead of blood) away from the lungs.

Lymph contains tissue fluid and waste
products, as well as immune cells.

Lung cancer cells can enter lymphatic
vessels and begin to grow in lymph nodes
around the bronchi and in the mediastinum
(the area between the 2 lungs).

When lung cancer cells have reached the
lymph nodes, they are more likely to have
spread to other organs. Treatment and
staging are in part based on whether the
cancer has spread to certain lymph nodes.
                      Types of Lung Cancer
Two major types of lung cancer:    Small cell lung cancer (SCLC)
                                   Non-small cell lung cancer (NSCLC)

(If both are present, it is called a mixed small cell/large cell carcinoma. (Rare)

SCLC and NSCLC generally require distinct treatments.

Small Cell Lung Cancer- ! 10% to 15% of all lung cancers are SCLC, named for
the small cells that make up these cancers. SCLC include oat cell carcinoma,
small cell undifferentiated carcinoma, & mixed small cell/large cell carcinoma.

SCLC often starts in the bronchi near the center of the chest, and tends to
quickly spread. The cells can multiply rapidly, form large tumors, and spread to
lymph nodes and other organs, such as the bones, brain, adrenal glands, & liver.

Surgery is rarely an option (and never the only treatment given). Treatment
includes chemotherapy to attenuate the widespread disease.

SCLC is frequently linked to smoking; it is rare in a non-smoker.
                   Types of Lung Cancer- NSCLC

Non-small Cell Lung Cancer- About 85% to 90% of lung cancers are non-small
cell lung cancer (NSCLC).

There are 3 major subtypes of NSCLC: adenocarcinoma, squamous cell
carcinoma, and large cell carcinoma.

The cells in these subtypes differ in size, shape, and biochemical composition
when examined microscopically.

Adenocarcinoma of the lung. This form of non-
small cell lung cancer (NSCLC) is now the most
common type. It typically starts in the
peripheral region of the lung.
                 Other Types of Lung Cancer
Besides SCLC and NSCLC, other tumors can occur in the lungs.

Carcinoid tumors account for < 5% of lung tumors. Most are slow-growing
tumors termed typical carcinoid tumors and are generally treated by surgery.

Although some typical carcinoid tumors can spread, they usually have a better
prognosis than SCLC or NSCLC. Atypical carcinoid tumors are less common and
the outlook for these tumors is between typical carcinoids and SCLC.

Other rare lung tumors include adenoid cystic carcinomas, hamartomas,
lymphomas, and sarcomas. Because the treatments for these tumors are
different from the more common lung cancers, they will not be discussed here.

Cancer that starts in other organs (breast, pancreas, kidney, or skin) and
metastasizes to the lungs is not the same as lung cancer. For example, breast
cancer that spreads to the lungs is still breast cancer (not lung cancer).

Treatment for metastatic cancer to the lungs depends on where it started (the
primary cancer site).
                   Lung Cancer- Statistics
Most of the values below include both SCLC and NSCLC (85-90% cases).

Lung cancer is the 2nd most common cancer in men (after prostate cancer)
and women (after breast cancer). It accounts for about 15% of all new
cancers. For 2008, ! 100,330 new cases (women); 114,690 new cases (men).

About 2 out of 3 people diagnosed with lung cancer are older than 65; fewer
than 3% are under 45. The average age at the time of diagnosis is about 70.

The chance of developing lung cancer is 1:16
(women) and 1:13 (men). For smokers, the
risk is #; for non-smokers, the risk is $.

The rate of lung cancer is $ among men and
is fairly stable among women.

Despite the very serious prognosis of lung
cancer, some people are cured. More than
400,000 people alive today have been
diagnosed with lung cancer at some point.
           Lung Cancer- Selected Risk Factors I
Tobacco Smoke- leading risk factor for lung cancer (especially SCLC). Certain
people are more susceptible to the cancer-causing effect of tobacco smoke;
this is in addition to those that have genetic predispositions.

Radon- Naturally-occurring radioactive gas that forms from the breakdown of
uranium in soil and rocks. According to the EPA, radon is the 2nd leading
cause of lung cancer, and is the leading cause among nonsmokers.

Asbestos- Workplace exposure to asbestos fibers is an important risk factor
for lung cancer. Both smokers and nonsmokers exposed to asbestos also have a
greater risk of developing mesothelioma, a type of cancer that starts in the
pleura (the lining surrounding the lungs).

Other Cancer-causing Agents-
   Radioactive ores such as uranium
   Inhaled chemicals or minerals such as beryllium, cadmium, vinyl chloride,
       Ni or Cr compounds, coal products, mustard gas & chloromethyl ethers
   Diesel exhaust

Dietary Factors (diet rich in fruits and vegetables may be beneficial)
        Lung Cancer- Selected Risk Factors II
Radiation Therapy to the Lungs- People who have had radiation therapy to the
chest for cancer (Hodgkin disease or women who have radiation therapy after a
mastectomy for breast cancer) are at higher risk for lung cancer.

Arsenic- High levels of arsenic in drinking water may # the risk of lung cancer.

Other Mineral Exposures- Silicosis and berylliosis are lung diseases caused by
breathing in certain minerals. People with these conditions also have # risk.

Personal and Family History of Lung Cancer- Not clear how much of this risk is
due to genetics vs shared exposures (tobacco smoke, radon).

Inherited genes appear to play a role in some families with a strong history of
lung cancer. People who inherit certain DNA changes, especially on chromosome
6, are more likely to develop lung cancer, even if they only smoke a little.

Air Pollution- In cities, air pollution (especially from heavily trafficked roads)
appears to raise the risk of lung cancer slightly.
          Lung Cancer- Molecular Mechanisms

Risk factors may produce certain changes in the DNA of cells in the lungs,
causing them to grow abnormally and to form cancers.

Certain genes control cell growth &
division. Genes that # cell division
and can cause cancer when elevated
or mutated are called oncogenes.

Genes that $ cell division or # cell
death at the appropriate time are
called tumor suppressor genes.

Cancers can be caused by DNA
mutations that turn on oncogenes or
turn off tumor suppressor genes.

Analysis of these systems is an actve
area of current study.
          Lung Cancer and Genetic Alterations
Inherited Gene Mutations- can greatly # the risk of developing certain cancers.
Markers on chromosome 6 (and possibly 12, 14, and 20) exhibit linkage to lung
cancer susceptibility. However, inherited mutations in oncogenes or tumor
suppressor genes are not thought to cause very many lung cancers.

Nonetheless, some people appear to inherit a $ ability to metabolize certain
types of cancer-causing chemicals, such as those found in tobacco smoke.

Other people may inherit faulty DNA repair mechanisms that make it more likely
they will end up with DNA changes. People with repair enzymes that do not work
properly might be vulnerable to cancer-causing chemicals & radiation.

Acquired Gene Mutations- those related to lung cancer usually develop during
life rather than before birth as an inherited mutation.

Acquired mutations in lung cells (via environmental agents) can appear in genes,
such as the p53 and Rb tumor suppressor genes, and may be important in the
development of SCLC. Changes in these and similar genes may also make some
lung cancers likely to grow and invade more rapidly than others.
           Lung Cancer-associated Genes
myc Family amplification first observed in SCLC.

                       Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
           Lung Cancer-associated Genes
ras Family genes linked with carcinogen (nitrosamine)-induced tumors.

                        Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
            Lung Cancer-associated Genes
EGF receptor overexpression/mutation in NSCLC.


                      Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
           Lung Cancer-associated Genes
ErbB2 (Neu, HER2) activation/overexpression in NSCLC.

                       Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
           Lung Cancer-associated Genes
Chromosome 3 deletion in SCLC- fragile histidine triad (FHIT) tumor

                        Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
           Lung Cancer-associated Genes
P53, Rb and APC in NSCLC and SCLC- tumor suppressors.

                      Hanahan and Weinberg (2000) The Hallmarks of Cancer. Cell 100, 57-70
                      Lung Cancer- Symptoms
Screening for Lung Cancer- Screening is the use of tests to detect disease prior to
symptoms; however, no lung cancer screening test has been shown to be effective
(although spiral or helical Computed Tomography (CT) has shown promise).

Signs and Symptoms- Most lung cancers do not cause any symptoms until late:

   *   A cough that does not go away; hoarseness; shortness of breath, wheezing
   *   Chest pain that is often worse with deep breathing, coughing, or laughing
   *   Weight loss and loss of appetite
   *   Bloody or rust-colored sputum (spit or phlegm)
   *   Recurring infections such as bronchitis and pneumonia

Lung cancer and/or its metastases may cause:

   * Bone pain
   * Neurologic changes (such as headache, weakness or numbness of a limb, dizziness, or
         recent onset of a seizure)
   * Jaundice (yellowing of the skin and eyes)
   * Lumps near the surface of the body, due to cancer spreading to the skin or to
         lymph nodes in the neck or above the collarbone
   * Endocrine disorders (SIADH, Cushing’s)

Note: These symptoms are more likely to be caused by conditions other than lung cancer.
          Lung Cancer- Imaging/Diagnostic Tests
*   Chest X-ray

•   Computed Tomography (CT): The CT (or CAT) scan uses X-rays to produce a detailed
    cross-sectional image. Recently, spiral (helical) CT has become available. CT can look
    for masses in the adrenal glands, brain & other organs, and to guide a biopsy needle.

*   Magnetic Resonance Imaging (MRI) : MRI scans provide a detailed image of soft
    tissues (using radio waves & strong magnets instead of X-rays). MRI scans are often
    used to evaluate possible spread of lung cancer to the brain or spinal cord.

* Positron Emission Tomography (PET): Involves
  injecting glucose (a sugar) containing a radioactive
  atom. Because cancer cells are growing rapidly, they
  absorb large amounts of sugar & a specialized
  camera creates a picture of its uptake in the body.

    Not as finely detailed as CT or MRI, but it can be
    important to see if the cancer has spread.

* Bone Scan- helps show if a cancer has metastasized
  the bones. Here, a small amount of radioactive
  material is injected & a specialized camera detects
  the radioactivity and creates a skeletal picture.
     Lung Cancer Diagnosis- Sampling Procedures
    Diagnosis is made by a microscopic examination of lung cells (cytology/histology). The
    cells can be obtained from lung secretions (phlegm) or by removing the cells (biopsy)
    from a suspicious area.

•   Sputum Cytology            Transbronchial lung-biopsy showing
                               subepithelial infiltration of small-cell
                               carcinoma (arrows). Hematoxylin–eosin
•   Fine Needle Biopsy (FNA)   staining; 40X and 400X.

•   Bronchoscopy (a lighted, flexible tube (bronchoscope) is passed through the mouth and
    down into the windpipe and bronchi.

•   Endobronchial Ultrasound (uses sound waves to create images; a bronchoscope is
    fitted with an ultrasound transducer at its tip and is passed down into the windpipe.
    (This test is not often done for SCLC.)

•   Endoscopic Esophageal Ultrasound (EUS) (involves using an endoscope (a lighted, flexible
    scope) that is passed down the throat and into the esophagus).

•   Mediastinoscopy (lighted scope) and Mediastinotomy (both involve surgical approaches)

•   Thoracentesis (needle sampling) and Thoracoscopy (lighted scope) to assess if cancer
    has spread to the space between the lungs and the chest wall, or to the linings.

     Lung Cancer- Other Tests and SCLC Staging
Other Tests-

Bone Marrow Aspiration and Biopsy: These tests are done to look for spread of the
cancer into the bone marrow.

Blood Tests- generally not used to diagnose lung cancer, but are often done to get a
sense of overall health and to help tell if cancer may have spread to other areas.

Pulmonary Function Tests- often performed after a lung cancer diagnosis to see how
well the lungs are working.

Small Cell Lung Cancer Staging Systems- Two staging systems are often used to
describe the extent of spread of SCLC:

Limited & Extensive Stage (helps with treatment decisions): Limited stage- the cancer is
only in one lung and perhaps lymph nodes on the same side of the chest. Extensive
stage- cancer spread to the other lung, lymph nodes on the other side of the chest, or
to distant organs. 2 out of 3 people with SCLC have extensive disease when diagnosed.

TNM Staging System: T indicates the size of the primary tumor and if it has grown into
nearby areas, N describes how much the cancer has spread to nearby (regional) lymph
nodes, and M indicates if the cancer has spread to other organs (liver, bones, & brain.)
                   Lung Cancer Treatment - SCLC
Radiation Therapy- uses high-energy rays (such as X-rays) or particles to kill cancer
   cells. External beam radiation therapy (EBRT) delivers radiation from outside the body
   that is focused on the cancer.

In SCLC, radiation therapy may be used in several situations:

•    Most often given at the same time as chemotherapy in limited stage disease to
    treat the tumor and lymph nodes in the chest. After chemotherapy, radiation
    therapy is sometimes used to kill any small deposits of cancer that may remain.

• It can be used to shrink tumors to palliate (relieve) symptoms of lung cancer such as
  bone pain, bleeding, trouble swallowing, cough, shortness of breath, and problems
  caused by brain metastases.

• It is often given to the brain in limited SCLC after other treatments to reduce the
  chances that the cancer will spread there. (Brain is a common site of metastasis.)

• Three-dimensional conformal radiation therapy (3D-CRT): 3D-CRT uses special
  computer programs to precisely map the location of the tumor(s). Radiation beams are
  shaped and aimed at the tumor(s) from several directions, which makes it less likely to
  damage normal tissues.
                Lung Cancer Treatment (Surgery)

Surgery- rarely used as the main form of treatment in SCLC

• If the entire lung is removed, the surgery is called a pneumonectomy.

• If a section (lobe) of the lung is removed, the operation is called a lobectomy
         (prefered for SCLC).

• Removing part of a lobe is known as a segmentectomy or wedge resection.

With these operations, lymph nodes are also removed assess possible cancer spread.

• Recently, a less invasive procedure for treating early stage lung cancer called video-
  assisted thoracic surgery (VATS) has also been used.

• Surgery to relieve symptoms of SCLC: In some cases, surgery may be used to help
  treat the symptoms of the cancer (as opposed to trying to remove all of the cancer).
                     Lung Cancer Treatment - SCLC
Chemotherapy- treatment with anti-cancer drugs injected into a vein or taken orally.
Chemotherapy for SCLC generally uses a combination of 2 drugs. The drug combinations
most often used for initial chemotherapy for SCLC are:

Limited Stage                                   Extensive Stage
* cisplatin and etoposide                        * cisplatin and etoposide
* carboplatin and etoposide                      * carboplatin and etoposide
                                                 * cisplatin and irinotecan

If the cancer progresses during treatment or returns after treatment, different drugs
may be tried. The choice depends to some extent on how soon the cancer begins to grow
again. (The longer it takes for the cancer to return, the more likely it is to respond.)

If the relapse occurs within 2 to 3 mo of finishing treatment, drugs such as topotecan,
ifosfamide, paclitaxel, docetaxel, or gemcitabine may be tried.

If the relapse occurs after 2-3 to 6 mo, topotecan is often used. Other drugs that may
be tried include irinotecan, cyclophosphamide/doxorubicin/vincristine (known as the CAV
regimen), gemcitabine, paclitaxel, docetaxel, oral etoposide, or vinorelbine.

For relapses 6 or more months after treatment, the original chemotherapy regimen may
still be effective and can often be tried again.
           Non-Small Cell Lung Cancer (NSCLC)
About 85% to 90% of all lung cancers are of the NSCLC type. There are 3 sub-types
of NSCLC. The cells in these sub-types differ in size, shape, and biochemical make-up.

Adenocarcinoma: This type accounts for about 40% of
lung cancers. Usually found in the outer part of the lung.

Squamous cell carcinoma: About 20% to 30% of all lung
cancers. Linked to smoking and tend to be found in
the middle of the lungs, near a bronchus.

Large-cell (undifferentiated) carcinoma: About 10% to 15%
of lung cancers. It can start in any part of the lung. Tends
to grow and spread quickly, which makes it harder to treat.

Others (carcinoid, etc.)

              NSCLC Chemotherapy @ 2007

Cisplatin and vinorelbine appear best
NEJM 2004;350:351, NEJM 2005;352:2589, Lancet Oncol 2006;7:719

Bevacizumab (anti-VEGF antibody) in advanced NSCLC-
frontline treatment in combination with chemotherapy
(taxol/carboplatin) E4599
(Sandler et al. NEJM 2006;355:2542)

–Response rate 27% vs. 10%

             NSCLC Treatment and EGF

Epidermal growth factor receptor (EGFR) inhibition in advanced NSCLC

– 10% response rate in advanced disease, 30% prolonged stabilization
– Survival advantage (erlotinib)
    • Shepherd, F. A. et al. N Engl J Med 2005;353:123-132
– Mutually exclusive with K-ras
– Most benefit for non-smoking related NSCLC, with EGFR mutations
  (females, adenocarcinomas), but benefit in non-mutated as well
    • Lynch et al., NEJM 350:2129, 2004; Paez et al., Science
      304:1497, 2004; Pao et al., PNAS 101:13306, 2004
– Mechanisms of secondary resistance to EGFR inhibitors being identified
  (T790M mutation-50%, Met amplification-20%)
    • Pao et al., PLoS Med 2:e17, 2005; Engelman et al., Science
      316:1039, 2007

                Other NSCLC Treatment Options

Surgery- usually recommended (often along with other treatments) for early stage lung
cancers. If surgery can be done, it gives the best chance of curing NSCLC.

Many different operations can be used to treat (and maybe cure) NSCLC:
Pneumonectomy, lobectomy, or segmentectomy or wedge resection. Also, (for early stage
lung cancer) - video-assisted thoracic surgery (VATS).

Radiation Therapy- sometimes used as the main treatment of lung cancer. Can be used for
people who are not healthy enough to have surgery, to kill small areas of cancer, to relieve
symptoms. A special kind of radiation (called the Gamma Knife®) can sometimes be used
instead of surgery for single tumors that have spread to the brain.

Radiofrequency Ablation (RFA)- an electric current is passed through a probe, which heats
the tumor and destroys the cancer cells.

Photodynamic Therapy (PDT)- sometimes used to treat smaller lung cancers near airways,
or to help open up airways blocked by tumors to help people breathe better.

Chemotherapy, Vaccines, and Targeted Therapies using drugs that block tumor blood vessel
growth (angiogenesis) such as Avastin®, or that target the receptors for growth factors
such as EGFR (Tarceva/Erlotinib).
Cancer Cell Biology and the Epidermal Growth
               Factor Receptor
                   Epidermal Growth Factor

• Peptide hormone generated from a large
  transmembrane precursor by several tissues.

• Regulates     the    proliferation,    survival,
  differentiation, and/or motility of many cells
  (e.g., fibroblasts & epithelial cells).

• Affects diverse processes such as wound
  repair, adipogenesis, muscle contraction,
  neurite outgrowth and pituitary function.

• Binds to a 170 kDa transmembrane receptor
  (EGFR) that possesses intrinsic ligand-
  stimulated tyrosine kinase activity.
 Epidermal Growth Factor Receptor (EGFR)

                Transmembrane and
Cysteine-Rich                        CAIN
              Juxtamembrane Domains
  Domains                           Domain

                                                      Y1086 Y1173

                                            Y992 Y1068 Y1148

Ligand Binding        Tyrosine Kinase          C-terminal Tail
Domain (LBD)              Domain

   The EGFR is the cellular homolog of v-Erb, which is a viral
       gene product of the Avian Erythroblastosis Virus-
                 Erythroleukemia and Sarcoma
       Epidermal Growth Factor Receptor-
             A Cell Signaling Nexus

           Multiple Ligands          Tyrosine Kinases

Interacts with: Other Hormone and Growth Factor Receptors (PDGF)
                Cytokine Receptors (Growth Hormone, Prolactin)
                Extracellular Matrix Receptors (Integrins)
                G-Protein Coupled Receptors (LPA, Thrombin)
                Other EGF Receptor Family Members
“Textbook” EGF Receptor Signaling and
 Regulation of MAP Kinase Activation

                              Enhanced        Small MW G-
                            Tyrosine Kinase     Protein
      Homo- and Hetero-
     Dimerization of ErbB                      MAPKKK
       Family Members


      Multiple Isoforms of                      MAPK

   Adapted from Sigma-Aldrich
    Selected Effectors of the EGF Receptor

         EGF           EGF

                              PLC%                 DAG
           ATP         ATP
                                            IP3            PKC
                 PP    P
     Shc         PP    P                            & Ca++
            P                   & Growth
                 P     P
                                & Differentiaton
                                & Motility
                           STATs 3, 5
Effects of EGF Receptor Stimulation in
            Tumor Biology

• Increased proliferation (cell cycle progression)
• Metastasis and migration
   – Altered regulation of cell adhesion and motility
• Angiogenesis and neovascularization
• Increased invasiveness
• Inhibition of apoptosis
                EGF Receptor in Cancer




*Overexpression                         *Kinase Domain Mutations
   (e.g., head and neck)                   (e.g., lung cancers)

   *Activating Deletions               Activating Insertions
      (e.g., brain)                       (e.g., brain)
                           *Citri et al. (2006), Nature Reviews Molecular Cell Biology.
    The EGF Receptor As A Key Therapeutic Target

!   Dysregulation of the EGFR has been linked to many malignancies.
     ! Ligand over-production
     ! Over-expression of WT EGFR
     ! EGFR mutations
                                                           Rowinsky et al, 2004, Annu. Rev. Med.

!   Lung Cancer accounts for more mortality each year than breast, prostate,
    and colon cancer combined.
     ! > 170,000 people are diagnosed with lung cancer each year.

                                                           Jemal et al, 2007, CA Cancer J Clin

!   Recently EGF receptor mutations have been identified in lung cancer patients,
    Non-Small Cell Lung Cancer (NSCLC), that responded to a chemotherapeutic
    agent selective for the EGFR (e.g., Gefitinib).
         Pao et al, 2004, PNAS;   Lynch et al, 2004, N Engl J Med;   Paez et al, 2004, Science
Potential Therapy for NSCLC: Gefitinib and Other
            EGF Receptor Antagonists
!   Gefitinib (Iressa) is a potent inhibitor selective for the EGF receptor.
    ! Small-molecule tyrosine kinase inhibitor (4-anilinoquinazoline-based
      structure, similar to Erlotinib/Tarceva)
    ! Competitive inhibitor of ATP
    ! 100-fold selectivity compared to other tested kinases
    ! 1st in its class to be approved for clinical trials

!   In 2003, two Phase II Clinical trials treated random NSCLC patients
    with gefitinib.
    ! Positive response rates ranged from 9-19%
    ! However, later studies were less promising.

                                  Fukuoka et al, 2003, J. Clin. Oncol; Kris et al, 2003, JAMA
        EGFR Mutations Associated with NSCLC

•   Identified largely in Gefitinib-Sensitive NSCLC tumors
•   In tissue culture, when compared to wt EGFR, these mutants have been linked
    to :
     – " Cell proliferation.
     – " Phosphorylation of pAkt, pSTAT3, & pSTAT5

     Lynch et al, 2004, N Engl J Med; Sordella et al, 2004 Science
           EGFR and Gefitinib-Resistance
• Certain NSCLC patients that initially responded to
  Gefitinib became resistant to the drug.
   – These patients often harbored an additional T766M
     EGFR mutation.
                          Kobayashi S et al NEJM 2005, Pao W et al, PLOS Med 2005

                Gefitinib-Resistant EGFR Mutant
     EGF Receptor Tyrosine Kinase Domain





      Hypothesis: These EGFR mutations are associated with
altered/enhanced kinase activity (oncogenic capacity) but greater
     sensitivity to kinase antagonists (personalized medicine).
                                               Gefitinib-Inhibition of EGFR pY1068
       Relative to EGF/DMSO Control

                                                    125.00                                                                        WT (n=7)

                                                    100.00                                                                        !ELREA (n=4)

                                                     75.00                                                                        L837Q (n=3)

                                                     50.00                                                                        L834R (n=5)

                                                     25.00                                                                        L834R/T766M

                                      0         2            4               6             8             10             12

                                                                                    0 .0

                                                                                                                0 .1

                                                                                                                       0 .3
                                                                      0 .0

                                                                                           0 .0

                                                                                                  0 .0

                                                                                                         0 .0

                                                                             0 .0






                                                                                 Gefitinib (µM)
Basal, EGF-induced, and Gefitinib-Inhibition of Akt
       (Cell Survival Protein) Phosphorylation


             10 n



                            Gefitinib (µM)
Attenuated Tyrosine Kinase Activity of Purified
      !ELREA EGFR (NSCLC-associated)

                    Km                 Vmax            Vmax/Km
      EGFR    Substrate Affinity   Maximal Velocity   Overall Efficiency

       WT      0.69 +/- 0.12         2.1 +/- 0.2            3.0
                 1.3 +/- 0.2       0.98 +/- 0.07           0.75
     Conclusions Regarding NSCLC-Associated
                  EGFR Mutants
• NSCLC-associated EGFR mutants exhibit higher basal activation/

• Mutant EGFRs (single mutations) display greater sensitivity to
  kinase inhibitors (Gefitinib) compared to WT with respect to
  autophosphorylation and Akt activation/phosphorylation.

• The EGFR double mutant (T766M-containing) possesses Gefitinib-
  resistance (up to 1 µM Gefitinib).

• EGFR mutants appear to possess attenuated kinase activity
  perhaps allowing them greater sensitivity to Gefitinib antagonism
  (similar story is emerging with glioblastomas).

• The oncogenic capacity of the NSCLC-associated EGFR mutants
  may be linked to their # basal (chronic) EGFR activation status.
Molecular Analysis of EGF Receptor Expression
and Activation Using Nanostructured Surfaces
    and Liquid Crystal-Based Technologies

                    Paul J. Bertics
                  Nicholas L. Abbott

 Departments of Biomolecular Chemistry and Chemical &
                   Biological Engineering
            University of Wisconsin-Madison
                       General Background
•   Many potential anti-cancer agents target proteins that control cell cycle
    progression (growth), survival, or motility/adhesion.

•   One target is the epidermal growth factor receptor (EGFR) tyrosine kinase.

•   Certain EGFR mutations have been linked to tumor shrinkage following
    treatment with kinase inhibitors.

•   However, clinical trials with non-small cell lung cancer patients have
    revealed variable levels of effectiveness of kinase antagonists (Gefitinib).

•   Elucidation/validation of the precise mechanisms of action of these anti-
    cancer agents in human tumors is limited by the difficulty of performing
    relevant molecular assays on small amounts of tissue material.

•   Uncertainty about how these agents affect EGFR status/signaling in human
    tumors has influenced the clinical development of these drugs.

•   Need for rapid & cost effective methods for analyzing the
    expression, activation or inhibition of growth regulatory molecules.

•   Current methods are often time-consuming, laborious, modestly
    sensitive, expensive, utilize specialized equipment, etc.

•   Further develop a technology that uses nanostructured surfaces and
    liquid crystals (LCs) to amplify and image molecular interactions.

•   Preliminary data for this LC-based assay reveal that we can:

        a) Fabricate surfaces with nanometer-scale topographies
        b) Identify EGFR expression & phosphorylation in cell extracts
        c) Detect $ EGFR phosphorylation after exposure to EGFR
        Advantages of LC-Based Analytical Tools
1.   Extremely sensitive (103 events vs 105-108 events).

2. Can detect molecular binding events, enzyme activity & cell

3. Detects binding events within localized regions of a surface with mm
   resolution: Imaging of binding events can be highly multiplexed-

     "Snapshot" of the levels/modification of entire molecular

4. Optical outputs can be easily detected and quantified using visible

5. Rapid: Amplification (reorientation of LC) can occur in less than a
   second; whole assays in minutes.

6. Widely adaptable to the analysis of multiple macromolecules.
  General Concepts Associated with the
Development of Liquid Crystal-based Assays

(a) Structure of 4-cyano-4'-pentylbiphenyl, a chemical that acts as a
    liquid crystal at room temperature.

(b) Subtle changes at the surface are amplified into a change in the
    orientation of a thin (100 µm) film of liquid crystal.

(c) LCs that can be used for amplification and transduction of events
    of at the surface of a sensor.
      Liquid Crystal-Based Technology Can
      Report the Specific Binding of Proteins
                 (Antibodies, Ab)

                 No Ab           Anti-Biotin Ab      Non-specific Ab

• Immobilize biotin to the gold surface followed by incubation for 15 min with
    various Antibodies (Abs).

• Add Liquid Crystal and measure light transmission.

• Record images.
     Stamping Approach for the Detection of
Biomolecules Using Nanostructured Surfaces & LCs
    Polydimethylsiloxane Stamp          Incubate with Extract and Wash


              of Capture
          of anti-EGFR                            Printing

                                                Amine SAM
                                         Nanostructured Gold
                                                                            Evaluate using Optical Output

                           Add Liquid Crystal                      Liquid Crystal

 (SAM=self-assembled monolayer)
General Strategy of EGF Receptor Analysis
      Using LC-Based Technology
                  Pan-reactive                                          Anti-active antibody

                                         nanostructured surface

        pan-reactive   anti-active        pan -reactive   anti-active      pan-reactive   anti-active

                             increasing extent of activation of EGFR

                                     Total EGFR

    Gray -scale
    Of LC                                                     Autophosphorylated EGFR

                          Extent of autophosphorylation of EGFR
•   Need for rapid & cost effective methods for analyzing the expression,
    activation or inhibition of growth regulatory molecules.

•   Current methods are often time-consuming, laborious, modestly sensitive,
    expensive, utilize specialized equipment, etc.

•   Further develop a technology that uses nanostructured surfaces and liquid
    crystals (LCs) to amplify and image molecular interactions.

•   Preliminary data for this LC-based assay reveal that we can:

         a) Fabricate surfaces with nanometer-scale topographies
         b) Identify EGFR expression & phosphorylation in cell extracts
         c) Detect $ EGFR phosphorylation after exposure to EGFR antagonists

•   Future Goals: Refine, validate, & implement this approach for high
    throughput screening of limited clinical specimens, and to ascertain which
    tumors are most likely to respond to EGFR antagonists.
Biomolecular Chemistry           Chemical and Biological Engineering
     Paul J. Bertics                     Nicholas L. Abbott
   Gregory J. Wiepz                        Aaron M. Lowe
    Byram H. Ozer                            Yiqun Bai
   Chimera R. Peet                    Govindaraja Thimmaiah
  Nicole Korpi-Steiner                   Chang-Hyun Jang
                                         Matthew L. Tingey
  Arturo Guadarrama

                         Ronald T. Raines
                            Jeet Kalia

                          Eric V. Shusta
                          Nitin Agarwal

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