MOLECULAR BIOLOGY

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MOLECULAR BIOLOGY PATHOLOGY IN EPIDEMIOLOGY
JianYu Rao, M.D. Associate professor of pathology
and epidemiology UCLA
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Molecular Biology - Outline

• Introduction
• Basic Principles of Molecular Biology
• Core Techniques of Molecular Biology
• High Throughput Technologies
• Epigenetics DNA Methylation

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INTRODUCTION

• 1953 - Discovery of DNA double helix (Crick
  Watson)
• 1960s - DNA transcription mechanism
• 1970s - Recombinant DNA technology
• 1980s - PCR
• 1990s - Human genome project/DNA chips
• 2000 Genome Wide Association (GWA) Studies

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Basic Principles of Molecular Biology

• DNA structure
• 4 bases (nucleotide) 2 pyrimidines thymine (T)
  and cytosine (C), and 2 purines adenine (A) and
  guanine (G)
• Form double helix by base-paring through H-bond
  (A to T and G to C) and a backbone consists of
  sugars and phosphate.
• The strands have polarity (3 to 5 or vice
  versa) and are complementary to each other.

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• Genetic information is organized lineally
• A codon is the basic unit with 3 consecutive
  nucleotides that specifies a single aa.
• A gene is a segment of DNA (with lineally linked
  multiple codeons) that specifies a protein.
• A chromosome contains several thousands genes and
  is the smallest replicating unit (human has 46
  chromosomes).
• The genome is the entire set of information that
  an organism contains.

5 3
5' CCT GGT CCT CTG ACT GCT - 3'
K H L
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Basic Principles of Molecular Biology (cont.)

• Gene structure
• Gene is compose of a upstream 5 regulatory
  region (TATA box or CAAT box), several exons
  (expressed gene sequences), and intervening
  intrones (nonexpressed sequence).
• There are a total of 100,000 genes estimated in
  mammalian genome.
• Less than 30 of the genome is ever transcribed
  into RNA, and only a fraction of that is
  translated into protein.

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• More than 70 of entire genome is not transcribed
  and is composed of many stretches of repetitious
  sequences that can repeat on scales of 5-10 bp,
  to 5000-6000 bp. Species specific type of
  repeats, termed Alu sequences, are useful as
  markers for identifying genes transferred between
  species.
• A gene family are a number of closely linked
  genes that code for structurally and functionally
  related proteins.

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Basic Principles of Molecular Biology (Cont.)

• Gene transcription (DNA to mRNA)
• mRNA (message RNA) is the template for protein
  synthesis.
• Only the exon sequences of a given gene is
  transcribed.
• Transcription begins by binding of RNA polymerase
  II on initiation site. This process requires a
  transcription factor which is a protein
  recognizing the region of DNA to be transcribed.
  

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• A primary transcript which ranges from the
  initiation site to a termination site (including
  all the exons and introns) is produced initially,
  followed by adding a cap (methylated G) at 5 end
  and a Poly A tail at 3end, and finally by
  several steps of splicing (cut off the introns).
• The produced mature mRNA is then exported from
  nuclear to cytoplasm by unknown mechanisms for
  translation.

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Basic Principles of Molecular Biology (Cont.)

• Translation (mRNA to protein)
• The translation is taken place in cytoplasm, in
  ribosomes.
• Proteins are further modified by
  post-translational modification steps, including
  proteolytic cleavage, addition of carbohydrate or
  lipid motifs, and modification of a.a..
• Gene expression in a cell is influenced by both
  the micro (surrounding cell, tissue, organ) and
  macro (endocrine and paracrine) environments.

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Core Techniques

• Restriction Endonucleases
• Enzymes found in bacteria that cleave DNA at
  precise sequences.
• Named by the organisms of origin (eg. EcoRI is
  from E Coli R strain).
• Size of fragments produced is a function of the
  number of the bases in the restriction site.
  (eg., 4 cutters produce DNA into smaller
  fragments while 8 cutters produce gene-sized DNA
  fragments).

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Core Techniques (Cont.)

• Hybridization
• Based on the property of DNA base paring (A to T
  and G to C).
• The principle is the recognition of a
  complementary sequence (gene to be detected) by a
  short sequence (Probe) .
• The two strands of targeted DNA needs to be
  separated into single strands by a process of
  melting at first, followed by annealing (reform
  the double strand) after adding the probe.

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• The annealing depends on several factors,
  including DNA concentration, the time, the
  temperature, and the concentration of salts. The
  stringency of annealing is a function of
  temperature and salt concentration.
• Examples
• Dot or slot blot
• In situ hybridization (FISH, gene or chromosome)
• Northern or Southern blot
• Needs to know the DNA sequence to be fished.

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Core Techniques (Cont.)

• Electrophoresis
• A technique to separate nucleic acids and
  proteins by size and charge.
• All electrophoretic techniques are carried out
  using a supporting gel of controlled pore size.
• Most separations are by size of moleculars (large
  one stay, the small one migrate), while the
  charge governs the actual migration of the
  moleculars.
• Polyacrylamide - for small noncharged moleculars
  (DNA)
• Agarose - for large noncharged moleculars
  (DNA/RNA)
• urea and SDS - for charged moleculars (protein)

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• Procedure
• Making a gel and buffers (loading and running
  buffers)
• Apply sample into the well
• Apply voltage (100 to 1000s depends on the size
  of gel)
• Visualize and detection (staining the gel, or
  transfer the moleculars into membranes)

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Core Techniques (Cont.)

• Sourthern blot - for DNA (RFLP)
• Northern blot - for RNA
• Western blot - for protein

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Core Techniques (Cont.)

• Isolation of DNA and RNA
• It is crucial to have pure source of DNA or RNA
  for the accurate analysis.
• The purity is indicated by the ratio of OD
  reading (OD 260 vs 280, which measures nucleic
  acids vs protein, respectively)
• RNA is much less stable than DNA, due to the
  widely present RNases.
• The major method for DNA isolation is the
  phenol-chloroform extraction (phenol allows
  dissociation of DNA from protein, whereas
  chloroform promotes the protein denaturation).
  Followed by separation with centrifugation, the
  DNA is present at upper phase.

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• The major method for mRNA isolation is by
  modified phenol-chloroform method that requires a
  inhibition of RNase using guanidinium and a
  purification step using either oligo(dT)
  chromatography or beads.
• Source of DNA can be any fresh or archived small
  amount materials (paraffin blocks, trace amount
  of old blood, saliva, etc), while mRNA usually
  requires large amounts of fresh or immediately
  frozen samples.

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Core Techniques (Cont.)

• PCR (Polymerase Chain Reaction)
• Revolutionize the detection technique for nucleic
  acids (DNA and RNA), also useful for cloning and
  site-directed mutagenesis.
• The principle is by cycling the temperature
  changes from denaturation (95 C), annealing
  (50C), and hybridization (70C), it allows a
  molecular (single stranded) to replicate itself
  exponentially.
• Requires primers, DNA polymerase, nucleoside
  triphosphates, and magnesium ion.

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• Limitations of PCR
• Primer selectivity
• Primer dimer formation
• Contamination
• Nonspecific priming
• Temperature design for GC rich or AT rich genes
  (incomplete melting or incomplete annealing,
  respectively).
• In epidemiological studies it is used for
  detecting the presence/absence of genes (DNA or
  RNA), measures the level of genes, or detect the
  specific forms of mutations, etc.

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Core Techniques (Cont.)

• Examples of Variant PCR
• LCR (for detection of point mutation)
• Competitive PCR (for quantification of DNA copy
  )
• RT-PCR (for mRNA detection and quantification)
• SSCP (for screening of gene mutation)
• In situ PCR
• TRAP (for telomerase activity detection)
• Real-Time PCR

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Core Techniques (Cont.)

• Monoclonal Antibodies
• Or so called immunoglobulins, are antibodies
  capable of recognizing only one specific antigen
  (epitope).
• Developed by various techniques e.g., hybridoma,
  Phgae-display, etc.
• Used in molecular epidemiological studies to
  detect any protein products (such as oncogene
  products, growth factors, receptors, etc) in a
  highly specific and often quantitative manner by
  various methods such as ELISA, EIA,
  immunohistochemistry, immunocytochemistry, etc.

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• All these methods are basically use the same
  principle, i.e.,antigen-antibody reaction. They
  can be either direct (without amplification step)
  or indirect (with amplification steps)and a
  detection step (with enzyme colormatrix or
  fluorescence).
• 3 steps immunofluorescence to detect a tumor
  specific antigen M344
• Step 1 Incubate cells with McAb (mouse anti
  human) against M344
• Step 2 Incubate with biotinlated Goat (or
  rabbit) anti mouse IgG (amplification)
• Step 3 Incubate with streptavidin-Texas Red
  (amplification/detection)

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QFIABiomarker Profile
G-actin Texas-Red conjugated DNase I M344
FITC (or Rhodamin) 3- Step Immunofluorescence
DNA Hoechst or DAPI
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Core Techniques (Cont.)

• RFLP - Microsattelite marker - SNP
• RFLP is the method to detect alterations
  (mutation) of one specific gene.
• Microsattelite markers are simple tandem repeat
  polymorphisms of several locus, which replaces
  RFLP as markers for disease
• SNP - are single nucleotide variants of entire
  genome - therefore are much more powerful and may
  replace Microsattelite markers or RFLP as markers
  of disease
• More prevalent in the genome than microsattelites
  in genome
• Some SNPs located in genes directly affect
  protein structure or expression levels
• More stably inherited
• Better for high throughput analysis

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SNPs - Definition

• Single base pair positions in genomic DNA at
  which different sequence alternatives (alleles)
  exist in normal individuals in some population,
  wherein the least frequent allele has an
  abundance of 1 or greater (Brookes, Gene,
  1999).

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How to Define SNPS?

• Conventional way
• develop sequence tagged sites (STS)
• identify DNA sequence variants
• estimate allele frequencies of the marker
• place the marker in human genome
• obtain DNA sequence
• More powerful Genome Wide Association Studies
  (GWA)

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Genome Wide Association (GWA) Study

• Help to identified genetic susceptibility markers
  for cancer
• Prostate Chromosome 8q24 (Gudundsson, et al,
  Nature genetics/Yeager, et al, Nature Genetics,
  2007)
• Lung Chromosome 15q25 (nicotinic acetylcholine
  receptor subunits) (Huang, et al, Nature
  2008/Amos, et al, Nature Genetics,
  2008/Thorgerisson, et al, Nature genetic, 2008)
• Genes identified in these locus may also be the
  targets for chemopreventive drug development

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High Throughput Techniques

• Microarray technology
• DNA chips
• cDNA array format
• in situ synthesized oligonucleotide format
  (Affymetrix)
• Proteomics
• Tissue arrays
• These are powerful tools and high through put
  methods to study gene expression, but they are
  not the answers themselves
• Individual targets/patterns identified need to be
  validated
• In epidemiological studies, these methods can be
  used to identify specific exposure induced
  molecular changes, individual risk assessments,
  etc.

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Proteomics

• Examine protein level expression in a high
  throughput manner
• Used to identify protein markers/patterns
  associated with disease/function
• Different formats
• SELDI-TOF (laser desorption ionization
  time-of-flight) the protein-chip arrays, the
  mass analyzer, and the data-analysis software
• 2D Page coupled with MALDI-TOF (matrix-assisted
  laser desorption ionization time-of-flight)
• Antibody based formats

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Fig 1
A, GTE (20?g/ml)
pI
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MW (kDa)
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B, GTE (40?g/ml)
pI
20
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MW (kDa)
1
1
10
5
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Time
48 hr
48 hr
24 hr
-


GTE
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Tissue Array

• Provide a new high-throughput tool for the study
  of gene dosage and protein expression patterns in
  a large number of individual tissues for rapid
  and comprehensive molecular profiling of cancer
  and other diseases, without exhausting limited
  tissue resources.
• A typical example of a tissue array application
  is in searching for oncogenes amplifications in
  vast tumor tissue panels. Large-scale studies
  involving tumors encompassing differing stages
  and grades of disease are necessary to more
  efficiently validate putative markers and
  ultimately correlate genotypes with phenotypes.
• Also applicable to any medical research
  discipline in which paraffin-embedded tissues are
  utilized, including structural, developmental,
  and metabolic studies.

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Bladder Array
Gelsolin
HE
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DNA Methylation

• DNA methylation plays an important role in normal
  cellular processes, including X chromosome
  inactivation, imprinting control and
  transcriptional regulation of genes
• It predominantly found on cytosine residues in
  CpG dinucleotide, CpG island, to producing
  5-Methylcytosine
• CpG islands frequently located in or around the
  transcription sites

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DNA Methylation (Contd)

• Aberrant DNA methylation are one of the most
  common features of human neoplasia
• Two major potential mechanisms for aberrant DNA
  methylation in tumor carcinogenesis

Silencing tumor suppressor genes (e.g. p16 gene)
Point mutation C to T transition (e.g. P53 gene)
SourceRoyal Society of Chemistry
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Promoter-Region Methylation

• Promoter-region CpG islands methylation
• Is rare in normal cells
• Occur virtually in every type of human neoplasm
• Associate with inappropriate transcriptional
  silence
• Early event in tumor progression
• In tumor suppressor genes
• Most of the tumor suppressor genes are
  under-methylated in normal cells but methylated
  in tumor cells. Methylation is often correlated
  with an decreasing level of gene expression and
  can be found in premalignant lesions

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DNA methyltransferases

• DNMTs catalyze the transfer of a methyl group
  (CH3) from S-adenosylmethionine (SAM) to the
  carbon-5 position of cytosine producing the
  5-methylcytosine
• There are several DNA methyltransferases had been
  discovered, including DNMT1, 3a, and 3b

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Pathology - Objective

• To learn basic histopathological terminology.
• To know different types of tumor.

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What is the difference between tumor vs
cancer

• Tumor Either benign or malignant
• Cancer Usually malignant

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Classification of Tumors

• Based on histological origin
• (epithelial, mesenchyme, etc..)
• Based on biological behavior
• (benign vs malignant)

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PATHOLOGICAL REPORT

• Tumor histological type.
• Tumor stage.
• Tumor grade.
• Other features (size, necrosis, lymphovascular
  invasion)

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CANCER HISTOLOGICAL TYPE

• Three Major Categories
• Epithelial Carcinoma
• Mesenchyme Sarcoma
• Hematopoitic Leukemia/Lymphoma
• Other Minor Categories
• Nevocytic Melanoma
• Germ cell Teratoma, Seminoma, Yolk sac tumor,
  Choriocarcinoma, etc
• Endocrine/Neuro Carcinoid/Insulinoma/small cell
  carcinoma, etc

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CARCINOMA

• Squamous Squamous Cell Carcinoma.
• Glandular - Adenocarcinoma.
• Transitional Transitional Cell Carcinoma.
• Small cell Small cell carcinoma

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SARCOMA

• Muscle
• Smooth muscle Leiomyosarcoma
• Skeletal muscle Rhabdomyosarcoma
• Fat Liposarcoma
• Skeleton Osteosarcoma
• Cartilage Chondrosarcoma

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Classification of tumor according to their
morphologic features (histology)

• Morphologic classification refers to the
  histologic classification made by pathologist
  based on microscopic examination.

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Benign vs Malignant Tumor

• The main distinction between benign and malignant
  tumor is
• Malignant tumor has invasion and metastatic
  potential whereas benign tumor does not.
• Malignant tumor has features of abnormal cellular
  differentiation whereas benign tumor usually not.

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Why histologic classification is important in
cancer epidemiology?

• Cancer is not ONE disease
• Different cancer types of same organ may have
  different exposure etiology, pathogenesis, as
  well as behavior, i.e., HETEROGENEITY

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Carcinoma

• Carcinoma (Cancer of the epithelium) 85
• Epithelium is the term applied to the cells that
  cover the external surface of the body or that
  line the internal cavities, plus those cells
  derived from the linings that form glands.

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Why most common cancers are epithelial origin?

• These cells are the first point of contact of the
  body with environmental substances, either
  directly (squamous cells) or indirectly
  (glandular cells).
• Epithelial cells usually have fast turn over
  rate, i.e., fast cell division, and their DNA can
  be damaged by carcinogens more often than
  non-dividing cells.

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Carcinoma Squamous cell

• Originates from stratified squamous epithelium of
  the skin, mouth, esophagus, and vagina, as well
  as from areas of squamous metaplasia, as in the
  bronchi or squamocolumnar junction of the uterine
  cervix. SCC is marked by the production of
  keratin.

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Skin Cancer
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Squamous Cell Carcinoma
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Carcinoma, Transitional Cell

• Transitional cell carcinoma - arise from the
  transitional cell epithelium of the urinary
  tract, such as bladder.

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transitional cell carcinoma of the urothelium is
shown here at low power to reveal the frond-like
papillary projections of the tumor above the
surface to the left. It is differentiated enough
to resemble urothelium, but is a mass. No
invasion to the right is seen at this point.
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TCC at high power
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Carcinoma Adenocarcinoma

• Adenocarcinoma - is carcinoma of glandular
  epithelium and includes malignant tumors of the
  gastrointestinal mucosa, endometrium, and
  pancreas and is often associated with
  desmoplasia, tumor-induced proliferation of
  non-neoplastic fibrous connective tissue,
  particularly in adenocarcinoma of the breast,
  pancreas, and prostate.

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Prostate Ca
Ovarian Ca
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Sarcoma

• Sarcoma is a malignant tumor of mesenchymal
  origin
• Sarcoma is often used with a prefix that denotes
  the tissue of origin of the tumor, as in
  osteosarcoma (bone), leiomyosarcoma (smooth
  muscle), rhabdomyosarcoma (skeletal muscle), and
  liposarcoma (fatty tissue).

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Classification of tumor according to stage
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Stage

• -is clinical assessment of the degree of
  localization or spread of the tumor.
• -generally correlated better with prognosis than
  dose histopathologic grading.
• -is examplified by the generalized TNM system,
  which evaluates size and extent of tumor (T),
  lymph node involvement (N), and metastasis (M).
• -different staging systems (WHO, TNM, etc),
  sometimes oriented toward specific tumors, e.g.,
  Dukes system for colorectal carcinomas.

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Classification of Tumor according to its
differentiation (grade)
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Grade of Disease

• Grading is histo-pathologic evaluation of the
  lesion based on the degree of cellular
  differentiation and nuclear features
•   Well Differentiated (Grade I)
• more resemble to normal tissue/cell
• Moderately differentiated (Grade II)
• - less resemblance of normal tissue/cell
• Undifferentiated (Grade III)
• - lost resemblance to normal tissue/cell

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Gleason's breakthrough was to develop a
reproducible description of the glandular
architecture, to which one assigns a score from 1
to 5. The pathologist looks for a major pattern
and a minor pattern to give a Gleason sum between
2 and 10. On the left is a picture adapted from
Gleason's 1977 article demonstrating the changes
in gland pattern as one goes from grade 1 to
grade 5 cancer. The glands in grade 1 cancer are
small and round. Grade 5 cancer is hardly forming
glands at all.
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Benign Prostate Hyperplasia
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Gleason Grade 1 Prostate Cancer
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Gleason Grade 1 Prostate Cancer
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At right is Gleason 3 CaP. The glands are
irregularly shaped. They are mixed in with some
normal glands. This tumor is infiltrating the
prostate.
At higher magnification, there are nests of
glands with no intervening stroma. This is
characteristic of higher grade CaP
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Here is Gleason 5, or poorly differentiated
cancer. You can see that it is invading the
seminal vesicle (stage T4)
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Precursors from intraepithelial neoplasia (IN)
to carcinoma in situ (CIS)
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NORMAL CIN 1 CIN 2
CIN 3
NORMAL LGSIL HG SIL
HGSIL
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• Important for Epidemiologist
•  
• Study nature history of disease progression
• Study genetic/environmental factors associate
  with disease progression
• Develop tools for risk assessment/early
  detection
• Targets for chemoprevention

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Additional Molecular Event
Exposure to Carcinogen
Precancerous Intraepithelial Lesions, (PIN,
CIN, PaIN..)
Cancer
Birth
Surrogate End Point Markers
Markers for Exposure
Markers of Effect
Tumor Markers
Genetic Suscep. Marker
CHEMOPREVENTION
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SUMMARY

• The key is to understand tumor hetergeneity
• Human cancer is not one disease , but many
  different types of diseases (Disease
  heterogeneity).
• The same type/stage/grade of tumor may behave
  differently in different person (Behavior
  heterogeneity).
• Even within the same tumor, there are may be
  different histological appearances and molecular
  expressions/changes (Expression heterogeneity).
• As an epidemiologist, we should know the basic
  features of the disease, and design studies
  accordingly

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Thank You!
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Thank You!!