Molecular Pathology

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Molecular Pathology

• Carleton T. Garrett, MD, PhD
• Friday - August 16, 2002
• Department of Pathology
• Medical College of Virginia/VCUHS

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How do youDIAGNOSEDISEASE?
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Find a TEST that is POSITIVE if the person HAS
DISEASEANDNEGATIVE if the person doesNOT HAVE
DISEASE(perfect test)
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UnfortunatelyThere is no such thing as a
Perfect Test
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Clinical Sensitivity of a test

• Clinical Sensitivity is the likelihood
  (probability) that a test will be POSITIVE if a
  person HAS disease .

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Clinical Specificity of a test

• Clinical Specificity is the likelihood
  (probability) that a test will be NEGATIVE if a
  person does NOT HAVE disease.

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The feature that is measured in a clinical test
is referred to as an (bio)markeroranalyte
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Test to determine if a patient has been bitten
by a tickbiomarker gt RASH
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Rash
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Black-legged (deer) ticks
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RASH is a Sensitive Clinical Marker of a tick
bite, that is, it is nearly always present in
patients with tick bites
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RASH is NOT a very Specific Clinical Marker of a
tick bite, that is, it is present in a lot of
patients who do NOT have tick bites
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The BIOMARKER that is measured in a clinical
laboratory test to diagnose a disease may be like
the RASH. It may be present in most patients with
the disease but it may also be present in many
patients without disease or who have a different
disease.
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Key Concept

• The Value of any Clinical Test is a Function of
  its Clinical Sensitivity and Specificity

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Clinical Sensitivity of a test is a function of
the ability of the test to detect the biomarker
(analytical sensitivity of the test)Clinical
Specificity of a test is a function of the tests
ability to ONLY DETECT the biomarker (analytical
specificity of the test)
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DNA (genomic RNA)

• Nature's Most Specific BioMarker for All Living
  Organisms

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Molecular Diagnostics

• Molecular Diagnostics is the branch of clinical
  laboratory testing which uses DNA and RNA as the
  biomarker for the clinical test

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Disease Prevalence

• Disease Prevalence represents the frequency with
  which a given disease is observed in a
  physicians patient population

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Predictive Value of a Test

• If the sensitivity and specificity of a test for
  a given disease are known and the prevalence of
  the disease is known then the probability that a
  patent with a positive test result has the
  disease can be calculated

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Clinical Use of DNA/RNA Testing

• Infectious Disease
• Neoplastic Disease
• Genetic Disease
• Identity Testing

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Clinical Tests that Detect DNA and RNASouthern
Hybridization AnalysisTarget Amplification
AssaysPolymerase Chain ReactionLygase Chain
ReactionTranscription Mediated Amplification
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Southern Hybridization Analysis
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DNAisdouble strandedmade up of 4 bases -
A,T,C,GThe bases are joined together through
phosphate diester bonds giving DNA a NEGATIVE
charge
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How are nucleotides linked in DNA?
The 5-O of one nucleotide is linked via A
phosphate group to the 3-O of the Next
nucleotide.
How the sugar is linked? 2-Deoxyribose is a five
carbon sugar that Is missing the OH at C-2. The
C-1 of the deoxiribose is connected to N-1 of
pyrimidine and N-9 of purine.
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Restriction Enzymes
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Restriction Enzyme Digestion of Genomic DNA
6 copies of the human genome from 3 cells E is
the restriction site for restriction enzyme
EcoR1 E 1 E 2 E 3 E 4 E
5 E 6 E
Probe
-
5 1 3 6 2 4
Separate the digested DNA fragments on a gel
using electrophoresis

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Overview Southern blot Analysis
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Transfer the DNA in the gel onto a membrane
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Denature the DNA on the membrane and hybridize a
probe to the DNA on the membrane to locate the
gene of interest
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The Probe is detected using some physical
property of a label which is attached to the
probe (frequently the probe is radioactive)
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Southern Hybridization Detects

• Presence (or Absence) of a Gene
• Variation in the sequence of a Gene
  (Polymorphism)
• Increased copy number of a Gene (Gene
  Amplification)
• Gene rearrangement (Chromosomal Translocation)

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Southern Hybridization Limitation

• requires that 10,000 or more copies of the target
  sequence (marker) be present in order for the
  test to be positive (limitation in test's
  sensitivity)
• can NOT detect point mutation

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Polymerase Chain Reaction (PCR)
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PCR Advantage

• easily creates 10,000 or more copies of the
  target sequence (marker) regardless of the number
  of copies originally present in the sample
  (increases test sensitivity)

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PCR
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PCR Amplification of a specific marker sequence
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CONTAMINATION

• Amplicon - copies of the marker (target sequence)
  that are created by the testing method ex.
  Fragments of DNA created by the PCR reaction.
• Template - copies of the marker (target sequence)
  that arise from natural sources ex. Aerosolized
  bacteria or cross contamination of a negative
  sample by a positive one.

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Measures to AVOID Amplicon Contamination

• Process specimens separately from where samples
  are amplified and analyzed.
• Control work flow - personnel move from specimen
  processing (low contamination) areas into sample
  analysis (high contamination) areas.
• Inactivate amplified product (degrade or
  cross-link it).
• Use an amplification method that relies on
  synthesis of RNA rather than DNA (RNA degrades by
  natural means more easily than DNA).

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Measures to AVOID Template Contamination

• Do NOT process specimens for infectious disease
  in same environment in which target organism is
  cultured.

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Other Types of Amplification Technology

• Ligase Chain Reaction (LCx - Abbott)
• uses 4 primers instead of 2
• requires ligation of adjacent primers to form
  product.
• Transcription Mediated Amplification (TMA -
  Gen-Probe)
• primary amplified product is RNA rather than DNA.

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Ligase Chain Reaction (Abbott Lab)
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Gap 6 base pairs
base
base
pairs
pairs
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Transcription Mediated Amplification (Gen-Probe)
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Micro Arrays

• Tests for the presence of a nucleic acid sequence
  by hybridizing a probe bound to a matrix to the
  target sequence.
• Many different probes can be bound to the same
  matrix.
• Therefore, a single sample can be evaluated for
  many different target sequences simultaneously.

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Micro Arrays

• Expression Arrays - tests for mRNA expressed in a
  tissue.
• Sequencing Arrays - tests for nucleotide sequence
  in a fragment of DNA (sequencing by hybridization
  - ideal for detection of single nucleotide
  polymorphismssnps).

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Micro Array
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Result from Micro Array Study
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Affimetrix Chip Device
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Classification of Diffuse Large B-Cell Lymphoma
by MicroarraysAlizadeh et al., Nature 403503,
3 Feb 2000
42 DLBCL 11 CLL 9 Follicular Lymphomas Samples of
normal and stimulated lymphocytes 17,856 human
gene arrays In all, 1.8 million measurements were
made on 96 normal and malignant lymphocyte
samples using 128 microarrays.
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Performing a Microarray Study
Extract RNA
Extract RNA
Normal
Tumor
Make cDNA Amplify by PCR
PCR Product Labeled with Green Dye
PCR Product Labeled with Red Dye
Mix
Hybridize on
Micro Array
Green Signal RNA Expressed in Normal Tissue
Red Signal RNA Expressed in Tumor Tissue
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Each Patient
List of Cases
Results for One Gene
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Clinically Distinct DLBCL Subgroups
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Advantages of Molecular Testing

• Known pathogens
• enhanced sensitivity and specificity
• improved turnaround
• New disease markers
• improved diagnosis and prognosis (ex. Molecular
  Staging)
• improved therapy (pharmacogenomics)

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Disadvantages of Molecular Testing

• Use of Complex 'Research' Procedures
• Labor Intensive
• High Cost

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Cancer is a disease of SOMATIC mutations that
alter gene expression in the cancer cellEither
the mutated gene or the mRNA or protein from the
abnormally expressed gene may serve as a
BIOMARKER for cancer
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Her-2/neu Amplification in Breast Cancer

• Breast cancer is a leading cause of cancer deaths
  in women.
• Her-2/neu encodes a growth factor receptor in
  breast epithelium. When this gene is over
  expressed in breast cancer cells, the patient is
  more likely to have recurrance of their disease
  and die.
• Survival in patients whose tumors over express
  Her-2/neu is improved by treating them with an
  antibody to Her-2/neu named Herceptin

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Detection of Increased Number of Her-2/neu Genes
by Fluorescence Insitu Hybridization (FISH)
Amplified
Normal
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HER2/neu Expression Evaluated by
Immunohistochemistry
Negative (overexpression negative)
1 () (overexpression negative)
2 () (overexpression positive)
3 () (overexpression positive)
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STAGE of Disease
The STAGE of a disease generally refers to the
degree of spread of the disease throughout the
body. The higher the stage the worse the
prognosis.
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Molecular Staging

• Identification of the extent of spread of a
  pathogen responsible for a disease under
  circumstances where the extent of spread can not
  be determined by conventional testing methods.
• Examples -minimal residual leukemia and other
  types of cancer, viral load

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Detecting tumor cells by detection of mutant
genes (DNA)
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Detection of t(1418) Translocation in Follicular
Lymphoma

• Detection by PCR utilizes primers to bcl-2
  oncogene on chromosome 18 and immunoglobulin gene
  on chromosome 14

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Follicular Lymphoma
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A treatment that sometimes cures patients with
lymphomas is to treat them with high (lethal)
doses of chemotherapy and then rescue them by
giving them back their own bone marrow.Patients
with lymphoma bearing a t1418 translocation who
underwent bone marrow transplant using their own
marrow taken during clinical remission had a
LOWER Survival if their bone marrow cells had PCR
detectable tumor cells even though no tumor cells
were detected using conventional light microscopy
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Detection of Tumor Cells by measuring the mRNA of
specific genes

• Detection of a NORMAL mRNA that is Expressed by
  the Tumor but not by the Target Tissue (Ex. Mucin
  or Cytokeratin which are Expressed by Breast
  Cancer Cells but not by Bone Marrow, Peripheral
  Blood or Lymph Node Tissue).
• Detection of CHIMERIC mRNA that is Only Expressed
  by Tumor Cells (bcr-abl in CML).

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PCR Amplification of a specific biomarker sequence
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Detection of t(922) in CML

• Detection of circulating blood cells expressing
  bcr-abl chimeric RNA at 6-12 mo following bone
  marrow transplant is an indicator of likely
  relapse.

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RT-PCR for bcr-abl
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RT-PCR for bcr-abl
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RT-PCR for bcr-abl in Post BMT Patients

• Study 346 patients, 3-192 mo follow-up, 634
  PB/BM
• Results
• 3 mo - PCR gt N.S.
• 6-12 mo - PCR gt Relapse (Plt.0001)
• 42 relapse with PCR vs 3 with -PCR
• Conclusion
• PCR _at_ 6-12 mo is independent predictor of
  relapse and provides opportunity for early
  intervention

Blood, 452632-8,'95 Radich et al
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Measurements of Viral Loads in Patients Blood
Can be Predictive of Disease Outcome
HIV HCV
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HIV Disease
HIV Virus
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HIV disease - Viral Load and Clinical Progression

• Quantitative RT-PCR necessary (antigenemia too
  low)
• Viral Load predicts progression to AIDS over 10
  year period
• gt10,200 copies/mlgt gt70 progress to AIDS die
• lt10,200 copies/mlgt lt30 progress to AIDS die
• HIV RNA levels - best predictor of long-term
  clinical outcome
• CD4 lymphocyte count - best predictor of
  immediate short term risk of developing a
  opportunistic disease

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HCV Infection in Chronic Hepatitis

• HCV is an RNA virus responsible for most non-HBV
  related chronic active hepatitis.
• HCV viral load is the earliest laboratory sign of
  viral infection and remains elevated in patients
  who develop HCV related chronic viral hepatitis.

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Fragile X Syndrome
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CGG Repeat in Fragile X Syndrome

• normal range 6-54 repeats
• premutation range 52-200 repeats
• full mutation range 200-gt1000 repeats
• alleles with gt200 repeats are hypermethylated,
  transcriptionally repressed

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Fragile X Clinical Features

• gt90 have mental retardation IQ 20-60, mean
  30-45
• in children, may present with hyperactivity,
  ADD, autistic features, hyperextensible joints,
  mitral prolapse
• after puberty macroorchidism, long face with
  large ears and prominent jaw

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FMR1 gene
EagI
EcoRI
EcoRI
CpG island
CGG repeat
2.8 kb
5.2 kb
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Human Identity Testing
DNA from 2 unrelated individuals is 99.95
identical Distinguishing between individuals
depends on identifying differences
(polymorphisms) in their DNA
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Types of DNA Polymorphisms

• Variable number of tandem repeats (VNTRs)
• core repeat sequence 8-50 nucleotides.
• highly informative (only 3-4 markers reqd).
• must use Southern Blot
• Microsatellites (Short Tandem Repeats STRs)
• core repeat sequence 2-8 nucleotides.
• less informative than VNTRs but easier to test 8
  microsatellites than 3 or 4 VNTRs.
• DNA may be partially degraded.
• Single Nucleotide Polymorphisms (SNPs)

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Microsatellites
Use PCR to amplify across the polymorphic
region (microsatellite) and separate fragments
using gel electrophoresis. Compare with markers
of known size.
caacaacaacaa
Primer2
Primer1
(p)
caacaa
Primer2
Primer1
(m)
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Monitoring Bone Marrow Engraphment
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Microsatellites
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Donor and Recipient

• Microsatellite analysis

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Paternity Testing

• If the tested man fails to possess in his DNA two
  or more of the alleles contributed to the child
  by the biological father then he is excluded.
• If a match is present then the probability of
  paternity is calculated.
• Microsatellite (STR) loci are a valid means of
  testing for paternity. Generally 8 loci are
  sufficient to obtain probabilities of paternity
  of gt99 in cases where the tested man is not
  excluded.

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MATCH
EXCLUSION
DNAmarker
DNAmarker
DNAmarker
DNAmarker
DNAmarker
DNAmarker
1 2 3 ..
1 2 3 ..
?
?
?
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Single Nucleotide Polymorphisms(SNPs)

• Most frequently found variation in human genome
  (1/Kb).
• May cause changes in drug metabolism.
• May change metabolism of key compounds which
  predisposes host to disease resistance or
  susceptibility.
• May be useful for human identity testing.

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Summary

• Nucleic acids, both from humans and pathogens,
  are being used increasingly as important
  biomarkers of disease in the clinical practice of
  medicine. This use is likely to only increase in
  the future.