Molecular Pathology

Molecular Pathology
Carleton T. Garrett, MD, PhD

Friday - August 16, 2002

Department of Pathology
Medical College of Virginia/VCUHS
How do you
DIAGNOSE
DISEASE?
Find a TEST that is POSITIVE if the person HAS DISEASE

AND
NEGATIVE if the person does
NOT HAVE DISEASE

(perfect test)
Unfortunately
There is no such thing as a ‘Perfect Test’
Clinical Sensitivity of a test
Clinical Sensitivity is the likelihood (probability) that a test will be POSITIVE if a person HAS disease .
Clinical Specificity of a test
Clinical Specificity is the likelihood (probability) that a test will be NEGATIVE if a person does NOT HAVE disease.
The feature that is measured in a clinical test is referred to as an

(bio)marker
or
analyte
“Test” to determine if a patient has been bitten by a tick

“biomarker” => RASH
Rash
Black-legged (deer) ticks
RASH is a Sensitive Clinical Marker of a tick bite, that is, it is nearly always present in patients with tick bites
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
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.
Key Concept
The Value of any Clinical Test is a Function of its Clinical Sensitivity and Specificity
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 test’s ability to ONLY DETECT the biomarker (analytical specificity of the test)
DNA (genomic RNA)
Nature`s Most Specific BioMarker for All Living Organisms
Molecular Diagnostics
Molecular Diagnostics is the branch of clinical laboratory testing which uses DNA and RNA as the biomarker for the clinical test
Disease Prevalence
Disease Prevalence represents the frequency with which a given disease is observed in a physician’s patient population
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
Clinical Use of DNA/RNA Testing
Infectious Disease
Neoplastic Disease
Genetic Disease
Identity Testing
Clinical Tests that Detect DNA and RNA

Southern Hybridization Analysis

Target Amplification Assays:
Polymerase Chain Reaction
Lygase Chain Reaction
Transcription Mediated Amplification
Southern Hybridization Analysis
DNAis:
double stranded
made up of 4 bases - A,T,C,G

The bases are joined together through phosphate diester bonds giving DNA a NEGATIVE charge
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.
Restriction Enzymes
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
Separate the digested DNA fragments on a gel using
electrophoresis
5
1
3
6
2
4
Probe
-
+
Overview

Southern blot Analysis
Transfer the DNA in the gel onto a membrane
Denature the DNA on the membrane and hybridize a probe to the DNA on the membrane to locate the gene of interest
The Probe is detected using some physical property of a label
which is attached to the probe (frequently the probe is
radioactive)
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)
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
Polymerase Chain Reaction (PCR)
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)
PCR
PCR Amplification of a specific marker sequence
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].
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).
Measures to AVOID Template Contamination
Do NOT process specimens for infectious disease in same environment in which target organism is cultured.
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.
Ligase Chain Reaction (Abbott Lab)
20
base
pairs
20
base
pairs
Gap
6 base
pairs
Transcription Mediated Amplification (Gen-Probe)
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.
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 polymorphisms[snps]).
Micro Array
Result from Micro Array Study
Affimetrix Chip Device
Classification of Diffuse Large B-Cell Lymphoma by Microarrays

Alizadeh et al., Nature 403:503, 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.
Performing a Microarray Study
Normal
Tumor
Extract
RNA
Extract
RNA
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
Each
Patient
Results for One Gene
List of Cases
Clinically Distinct DLBCL Subgroups
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)
Disadvantages of Molecular Testing
Use of Complex `Research` Procedures

Labor Intensive

High Cost
Cancer is a disease of SOMATIC mutations that alter gene expression in the cancer cell

Either the mutated gene or the mRNA or protein from the abnormally expressed gene may serve as a BIOMARKER for cancer
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
Detection of Increased Number of Her-2/neu Genes by Fluorescence Insitu Hybridization (FISH)
Normal
Amplified
Negative (overexpression negative)
1 (+) (overexpression negative)
2 (+) (overexpression positive)
3 (+) (overexpression positive)
HER2/neu Expression Evaluated by Immunohistochemistry
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.
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
Detecting tumor cells by detection of mutant genes (DNA)
Detection of t(14;18) Translocation in Follicular Lymphoma
Detection by PCR utilizes primers to bcl-2 oncogene on chromosome 18 and immunoglobulin gene on chromosome 14
Follicular Lymphoma
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 t14;18 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
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).
PCR Amplification of a specific biomarker sequence
Detection of t(9;22) 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.
RT-PCR for bcr-abl
RT-PCR for bcr-abl
RT-PCR for bcr-abl in Post BMT Patients
Study: 346 patients, 3-192 mo follow-up, 634 PB/BM
Results:
3 mo - +PCR => N.S.
6-12 mo - +PCR => Relapse (P<.0001)
[42% relapse with +PCR vs 3% with -PCR]

Conclusion:
+PCR @ 6-12 mo is independent predictor of relapse and provides opportunity for early intervention
Blood, 45:2632-8,`95 [Radich et al]
Measurements of Viral Loads in Patient’s Blood Can be Predictive of Disease Outcome
HIV
HCV
HIV Disease
HIV Virus
HIV disease - Viral Load and Clinical Progression
Quantitative RT-PCR necessary (antigenemia too low)
Viral Load predicts progression to AIDS over 10 year period
>10,200 copies/ml=> >70% progress to AIDS & die
<10,200 copies/ml=> <30% 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
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.
Fragile X Syndrome
CGG Repeat in Fragile X Syndrome
normal range: 6-54 repeats
premutation range: 52-200 repeats
full mutation range: 200->1000 repeats
alleles with >200 repeats are hypermethylated, transcriptionally repressed
Fragile X Clinical Features
>90% 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
FMR1 gene
CpG
island
EagI
EcoRI
EcoRI
CGG
repeat
2.8 kb
5.2 kb
Human Identity Testing
DNA from 2 unrelated individuals is 99.95% identical

Distinguishing between individuals depends on
identifying differences (polymorphisms) in their DNA
Types of DNA Polymorphisms
Variable number of tandem repeats (VNTRs)
core repeat sequence 8-50 nucleotides.
highly informative (only 3-4 markers req’d).
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)
Microsatellites
(p)
(m)
caacaacaacaa
Primer1
Primer2
caacaa
Primer1
Primer2
Use PCR to amplify across the polymorphic region
(microsatellite) and separate fragments using gel
electrophoresis. Compare with markers of known
size.
Monitoring Bone Marrow Engraphment
Microsatellites
Donor and Recipient
Microsatellite analysis
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 >99% in cases where the tested man is not excluded.
MATCH
EXCLUSION
DNAmarker
DNAmarker
DNAmarker
#1

#2

#3

#..
DNAmarker
DNAmarker
DNAmarker
#1

#2

#3

#..
?
?
?
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.
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.