2013

Introduction to
Molecular Diagnostics
THe eSSeNTiALS OF DiAgNOSTiCS SerieS
AdvaMedDx and DxInsights Present
INtroduction to Molecular Diagnostics

Abstract

Diagnostic tests are the foundation of Increasing automation is enabling

a successful health care system, sophisticated molecular tests to be
providing critical information that health performed in the full scope of health
care providers and patients need to make care settings, bringing state of the art
the right medical decisions. Diagnostics diagnostics to all areas of the world.
often provide objective, quantitative This report provides an overview of
measurements that inform every stage the current landscape for molecular
of care—prevention, detection, diagnosis, diagnostics, explains the key technologies
treatment, and successful management that are driving the molecular revolution,
of health conditions. illustrates the power of molecular
Molecular diagnostics is a dynamic diagnostics with some specific examples,
and transformative area of diagnostics, and concludes by noting several
leading to insights in research and challenges that have the potential to
treatment in many disease states that influence progress in this critical field
are revolutionizing health care. Molecular of medicine.
diagnostics detect and measure the
presence of genetic material or proteins
associated with a specific health condition
or disease, helping to uncover the
underlying mechanisms of disease and
enabling clinicians to tailor care at an
individual level – facilitating the practice
of “personalized medicine.”
Continuous innovation in technology
is increasing the speed and performance
of molecular diagnostics, and a future
in which whole genome sequencing
is routinely performed is not far away.

The Essentials of Diagnostics series: introduction 2

Table of Contents
Introduction to Molecular Diagnostics
The Essentials of Diagnostics Series

Introduction 2

Molecular Diagnostics 4

The Molecular Diagnostics Field 6

Molecular Biology – Genomics and Proteomics 7
Technology of Molecular Diagnostics 9
DNA Sequence Detection using Polymerase Chain Reaction (PCR) 10
The Clinical Role 12
Screening Test Example: HPV 13
Screening/Diagnostic Test Example: Cystic Fibrosis 13
Diagnostic Test Example: Chlamydia/Gonorrhea 14
Therapeutic Decision-Making Example: KRAS 14
Therapeutic Monitoring Example: HIV 15

The Future of Molecular Diagnostics 15

Conclusion 17
Acknowledgments 17
Glossary of Common Molecular Diagnostic Terms 18-19

The Essentials of Diagnostics series: Molecular Diagnostics 3

Introduction

Diagnostics are an integral and critical part of our provide critical insights at every stage of medical
health care system, as the results of these tests care—prevention, detection, diagnosis, treatment and
inform a wide range of medical decision making. From successful management of health conditions. Figure
the genetic tests that inform personalized cancer 1 illustrates the roles of diagnostics throughout this
treatment to the microbial culture that identifies the continuum of health care.
right antibiotic to fight an infection, diagnostic tests

Comprehensive Role of Diagnostics

Diagnostics can help clinicians optimally manage patients through the continuum of care.

Risk Screening Diagnosis Staging and Therapy Monitoring

Assessment Prognosis Selection

Description Diagnostic Applied to Use for Assess Used to Recurrence

tests to high-risk definitive severity predict monitoring
complement patient to diagnosis and/or risk efficacy Monotoring
traditional identify and general of recurrence or safety for treatment
risk factors disease typing Inform response efficacy
early adjuvent to specific
therapy treatments
decision
Clinical Implement Nip disease Refer to the Determine Do not waste Control disease
Implications wellness in the bud appropriate whether unproductive progression
programs with early specialist treatment is therapy with changes in
proactively treatment necessary treatment

SOURCE: DxInsights White Paper January 2012

Figure 1: Role of diagnostics through the continuum of health care.

Broadly speaking, two primary areas of health care While the traditional laboratory remains a mainstay
diagnostics are “in vivo” imaging and “in vitro” for diagnostic testing, significant testing is done
diagnostics, often referred to as IVDs. Imaging outside the laboratory, in such point of care settings
encompasses such technologies as X-Rays, ultrasonic as hospitals, physicians’ offices, and clinics, and for
waves, magnetic resonance, or radio-nuclear methods personnel in the field, such as emergency responders
that produce images of the body and its organs and and soldiers. Pregnancy tests and diabetes test
other structures. IVDs are tests performed on a strips are familiar examples of diagnostics that are
sample taken from the body (blood, tissue, sputum, available directly to consumers.
urine, etc.). “In vitro” is from Latin, meaning “on glass”,
The main categories of diagnostics are clinical
in reference to the glassware used to hold bodily
chemistry, immunology, hematology, microbiology
samples during culture or examination. Molecular
and molecular diagnostics. The diagnostics industry
diagnostics, the topic of this report, is a subset of in
continues to innovate in all of these important areas,
vitro diagnostics, and, therefore, the scope of this
and molecular diagnostics has captured particular
report is confined to this field, which is hereafter
attention in recent years because of the deep insights
referred to as “diagnostics” for the sake
these types of tests bring to diagnosis and treatment.
of convenience.

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Molecular Diagnostics
Molecular diagnostics is one of the most dynamic a specific gene mutation in their cancer that is
and transformative areas of diagnostics, leading associated with a specific type of lung cancer).
to advances in research and treatment that are
Of course, the ultimate power of personalized
revolutionizing health care across a wide range of
medicine is the ability to treat these smaller groups
diseases and health conditions.
with therapies tailored to the molecular profile of their
“Molecular diagnostics” is a broad term describing individual cancer. When specific proteins or genetic
a class of diagnostic tests that assess a person’s health sequences have a known association with a specific
literally at a molecular level, detecting and measuring health condition or disease, they are often referred
specific genetic sequences in deoxyribonucleic acid to as “biomarkers”” because they are markers of
(DNA) or ribonucleic acid (RNA) or the proteins they that condition or disease.
express. Molecular diagnostics identify gene, RNA,
Molecular diagnostics are the tools that are driving
and protein variations that shed light on whether
the continuing discovery of biomarkers at the research
a specific person is predisposed to have a disease,
level, which in turn leads to treatments designed
whether they actually have a disease, or whether a
around these biomarkers. Then molecular diagnostics
certain treatment option is likely to be effective for
play an additional critical role by ensuring that these
a specific disease. These tests also can detect and
new therapies are delivered to the right patients
quantify the presence of specific viruses, bacteria,
through more accurate diagnosis of the exact nature
or types of cells.
of their individual disease. This has led to the
Sequencing the entire human genome is a feat emerging field of companion diagnostics, in which a
that, when it was first accomplished by the Human molecular diagnostic test is used to identify whether
Genome Project, took an international, government- a specific therapy (a companion to the diagnostic)
led research consortium over 10 years and $2.7 is likely to be effective for an individual patient.
billion. (An initial draft of the entire genome sequence
Molecular diagnostic tests can help a woman
was published in 2001, ten years after the project
understand the likelihood that her breast cancer will
was initiated, and an essentially complete version
reoccur later in life, or tell a doctor what drug is the
was published in 2003.) Remarkably, molecular
right treatment for a late-stage melanoma patient.
diagnostics now can sequence a person’s entire
They can make it possible for couples considering a
genome for a cost that is rapidly declining and now
family to know if they are carriers of a cystic fibrosis
approaches $1,000.
gene mutation and therefore at risk of having a child
Many of our bodily processes, both normal and affected by cystic fibrosis. Molecular diagnostics
abnormal, as well as health or disease states, are can identify multiple strains of respiratory viruses in
driven by the interaction of our genes and the a single test, or monitor the level of HIV virus in a
proteins they produce that carry out specific patient’s blood to determine how well their treatment
functions within the body. Therefore, the ability is working. In these and many other ways, molecular
to quickly and accurately assess an individual’s diagnostics are transforming health care.
health at this molecular level is truly transforming
Molecular diagnostics today are routinely used in
the practice of medicine.
hospitals, reference labs, and blood banks. In the
The term “personalized medicine” has arisen from latter, molecular tests are used to screen donated
this field of molecular discovery. The term means that blood products for infectious diseases like hepatitis
understanding underlying molecular mechanisms and HIV. In hospitals, testing is often performed to
is empowering clinicians to evolve away from treating identify pathogens in patients with infections. In fact,
every patient based on what they have broadly in infectious diseases are one of the strongest growing
common with other patients (e.g., lung cancer) areas within the molecular diagnostics field. There
to treating them as individuals (e.g., a patient has is also increasing demand for such technologies at

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Molecular Diagnostics
the point of care (testing that occurs at the point of through 2017. Today, molecular diagnostics is
treatment or patient interaction with a health care employed primarily in developed markets (with the
provider, such as a doctor’s office or a clinic). United States and Western Europe accounting for
about 80%), where most of the five-year growth
There are far too many types of molecular diagnostic
is expected to take place.
tests to cite in this report, including thousands
of hereditary genetic tests, and the field is rapidly Currently, molecular diagnostic revenue is dominated
evolving. The report that follows provides an overview
of the current field for molecular diagnostics, explains
Global In Vitro Diagnostics Market
the technology that is driving the molecular revolution,
The global IVD market was about $45.6B in 2012.
illustrates the power of molecular diagnostics with
some specific examples, and concludes by noting Total IVD market
several challenges that may slow progress in this Molecular expected to grow at
about 7%
critical field of medicine.
Molecular market
The Molecular Diagnostics Field expected to grow at
about 12%
The overall global market for diagnostics was valued Rest of IVD Growth fueled by
at $45.6 billion in 2012 and is expected to grow at
infectious disease
about 7% annually over the next five years to reach and oncology testing,
a market size of $64.6 billion in 20171. The United and markets in new
States and Europe account for about 60% of that geographies
market, with Asia Pacific forecast as the highest SOURCE: Frost and Sullivan.
growth region over the next five years.
Figure 2: Molecular Diagnostics as a component in the global in vitro
The U.S. diagnostics market was about $15.5 billion in diagnostics market.
2012, with a forecasted growth rate of 5.8% over the
next five years. by infectious disease testing (50-60%), with about
another third attributable to genetic testing, and blood
In developing countries, growth is propelled largely
screening applications accounting for much of the
by increasing access (often meaning populations
rest. For the reasons discussed earlier in this report,
getting access to meaningful health care for the first
growth in the molecular diagnostics market is spurred
time) and increasing government investments in
by the excitement over its potential to revolutionize
health care. A recent example of a test that is meeting
areas across the value chain in health care.
this need is a new MTB/RIF assay that reduces the
time to diagnosis of tuberculosis/rifampin resistant For example, in pharmaceutical industry research,
TB from 8 weeks to 2 hours and can be done “in the understanding the mechanism of diseases is critical
field”, in a mobile van, powered by solar energy. While for discovering the next breakthrough targeted
there continues to be growth in developed markets as therapies. Identification of patients with a specific
well, it is tempered by a perfect storm of constrained genetic profile (hereditary or somatic) or signature
health care budgets, regulatory hurdles, and increasing that predicts response to a specific drug or dose is
evidence expectations from payers. essential to increasing the proportion of patients with
favorable outcomes, thereby enhancing the overall
Molecular diagnostics is currently a relatively small
effectiveness of the therapy in the treated population.
portion of the overall diagnostics market ($5 billion,
or about 11%), but the fastest growing segment Indeed, the FDA has stated that when a new therapy is
(See Figure 2). Globally, the molecular diagnostics developed for which there is a diagnostic that provides
market is estimated to grow over 12% annually essential information for its safe and effective use, it
will require concurrent approval or clearance of the
1
Market statistics from Frost and Sullivan.

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Molecular Diagnostics
diagnostic. It underscores that the development of that made Gram-positive cells absorb dye also made
these pharmaceuticals and companion diagnostics them more susceptible to certain antibiotics. The
is a key area of market growth. Gram stain gave diagnostics a new and prominent role
in guiding therapeutic decisions.
For pharmaceuticals already on the market, the
development of new companion diagnostics that The Gram stain remains a widely used diagnostic
can better guide the use of those pharmaceuticals is test due to its ability to quickly identify the presence
another area of growth. of infection, as well as aid doctors in making a quick
decision regarding antibiotic treatment. It remains only
a first-line tool, however, with significant limitations.
Molecular Biology – Genomics and Proteomics Today, cell cultures, additional antibiotic sensitivity
testing, and genetic information from molecular
The ability of a health care practitioner to efficiently diagnostics provide critical additional information.
deliver effective care depends upon their ability to
accurately identify the cause of the patient’s problem, With the advent of the field of molecular biology,
i.e., make the diagnosis. Over time, our understanding scientists began isolating and studying genes and
of the mechanistic basis of disease has increased biochemicals that serve as biomarkers in healthy
and furthered our ability to make more accurate and and diseased human cells and tissues. Our modern
specific diagnoses. microscopes and electron microscopes gave us
the ability to look at cells, their proteins, and other
Early healers needed only to feel the patient’s skin subcellular structures to see changes associated with
to diagnose “fever,” for example, but with further diseases. We began to understand disease resistance
understanding of different illnesses and their and immunity and why certain people developed
symptoms, doctors gained the ability to distinguish diseases and others didn’t.
between yellow fever, scarlet fever, bubonic fever
and other types of infections. But how do we learn about the mechanisms that are
the root causes of diseases? We must often go to the
There are records of the study of gross anatomy and molecular level to understand the many ways in which
pathology (organs and tissues) dating back to at diseases can develop within an individual. Our DNA is
least the ancient Greeks. It was the advent of modern the inherited material found in almost every cell of
microscopy and histopathology, or cellular pathology, the body, which governs the
in the nineteenth century, however, which enabled way our bodies develop
a tremendous leap forward in our understanding of and function.
the biology of disease at a cellular level. It became
possible to view infectious microorganisms in samples
We must DNA is composed
often go to the molecular
taken from the body, and it was further discovered that of two interlocking,
level to understand
the cells of some different types of microorganisms helical strands (the
the many ways in which
could be distinguished by whether or not they famous double
diseases can develop
absorbed certain dyes or stains. helix), each of
within an individual.
which is a string of
Initially, different stains were used to visualize different four chemical bases in
types of cells and bacterial structures to help both see varying sequence: adenine
and identify specific microorganisms. Danish scientist (A), guanine (G), cytosine (C),
Hans Christian Gram described this differential and thymine (T). Adenine on one strand is paired with
staining technique in 1884, and, in recognition of thymine on the opposite strand, and guanine is paired
his work, the terms Gram-negative (failure to stain) with cytosine, forming what are referred to as “base
and Gram-positive (stain absorbing) were coined to pairs”. Reference is often made to “nucleotides”, which
describe different categories of bacteria. Eventually, are the basic structural building blocks of DNA (and
it was realized that the same cell wall characteristics

The Essentials of Diagnostics series: Molecular Diagnostics 7

Molecular Diagnostics
RNA), consisting of a base combined with a sugar in the infectious agents themselves (e.g., viruses or
and a phosphate group. A strand of DNA, therefore, bacteria) that cause the disease.
is a chain of nucleotides. The terms “nucleotide” and
A particular gene whose progression of base pairs is
“base” are often used interchangeably in reference to
typical of the population at large is often termed the
pieces of a DNA sequence.
“wild type” or “normal” gene sequence. Gene variants
Each complete human DNA helix contains a sequence that are deviations from the wild type may or may
of about three billion base pairs. The sequence of not be clinically significant and extensive human
the bases is more than 99% identical in all people. research is often needed to determine their relevance.
The one percent of sequences that are variable is A gene variant present in more than one percent of
responsible for the differences that occur among the population is called a polymorphism and may be
humans. Our genes, which are the functional units considered a normal variant.
of DNA, are segments of the DNA strand that
There are three kinds of gene mutations, which may
range in size from a few hundred bases to more
be a change of only one base or a long sequence of
than 2 million bases.
bases. These are (1) hereditary – a mutation that
Genes produce RNA (ribonucleic acid) through a is passed from parent to child; (2) de novo – new
process called transcription, which in turn directs the mutations that arise in the egg or sperm cell, or shortly
production of the proteins that make up the machinery after fertilization, and so are repeated throughout the
of our cells—and, consequently, the structures and body; or (3) somatic – those that arise during life from
functions of our bodies. We refer to this protein environmental causes or through an error in DNA
production as the “expression” of the underlying replication, and are typically present in tumor cells.
genes. The complex specialization, or differentiation,
When specific mutations, or sets of mutations, are
of cells throughout the body is a result of only specific
known to be biomarkers associated with a disease or
genes being active in certain cells at certain times,
condition, molecular diagnostic tests can examine a
and therefore expressing proteins related only to
patient’s genes to determine whether those mutations
the functions of those cells. Differences in genes
are present. These tests may look only for those
among individuals, and the differential expression
certain gene variants, or map the entire sequence
of those genes in a given individual, account for the
of a targeted portion of DNA to detect all mutations
physiological diversity of our race, as well as many
in the sequence.
of our diseases and health conditions.
Depending on the nature of the biomarker in question,
An essential cellular function is DNA replication, in
molecular diagnostics therefore can assess a person’s
which the helix separates, each strand is duplicated,
risk of developing a disease, determine whether a
and then the duplicate strands combine through base
person is a carrier of a hereditary condition, screen for
pairing. This is how an exact copy of an individual’s
diseases that are present but not yet symptomatic, or
DNA is transferred from one cell to another during cell
provide a diagnosis of existing symptoms.
division so that every cell contains the same DNA.
The study of the activities and interactions of our
genes is referred to as genomics, and the related study
of protein activity is referred to as proteomics. In
these fields of study, molecular diagnostics examine
our DNA, RNA, and proteins to assess how they are
functioning. Diseases disrupt the function of cells
and, therefore, can be detected and/or monitored by
examining the molecular alterations of DNA, RNA, or
the proteins in patients’ tissues, fluids, or tumors, or

The Essentials of Diagnostics series: Molecular Diagnostics 8

Molecular Diagnostics
Figure 3: Molecular
diagnostics examine the
molecules in the cell,
i.e. the dna, rna or
proteins, and how their
role in human biology
and disease.

Technology of Molecular Diagnostics

a number of techniques are employed in modern diagnostics to detect and quantify specific dna or rna
sequences, as well as proteins. this report provides a summary overview of one of the most fundamental of those
techniques, the polymerase chain reaction, that is used to amplify specific sequences of dna or rna. subsequent
reports will elaborate on additional genetic test methods, such as in situ hybridization or whole genome
sequencing, and such protein detection tests as mass spectrometry. some of these additional technologies
are described briefly below:

• in situ hybridization (isH or FisH): a technique • Mass spectrometry (Ms): a technology that
that “unzips” dna or rna in the sample and uses determines the molecular mass of a charged
a so-called probe—a labeled dna or rna strand particle by measuring its mass-to-charge (m/z)
that hybridizes with the target, complementary ratio. this technology is used to find and analyze
sequence on an unzipped strand and thereby protein based biomarkers and is broadly classified
identifies and quantifies the target sequence into gel-based and gel-free techniques.
in the sample. FisH stands for Fluorescent in situ
• sequencing (ce, ngs): a technique used to map
Hybridization due to its use of a fluorescent probe
out the sequence of the nucleotides that comprise
that facilitates automated reading of the results.
a strand of dna. today, this can be done via
• chips and Microarrays: these technologies capillary electrophoresis (ce) or through multiple
simultaneously measure rna or cdna (the next generation sequencing (ngs) methods.
expression of a large number of genes), or dna
(single nucleotide polymorphisms (snPs), or
genome regions).

The essenTials of DiagnosTics series: Molecular diagnostics 9

Molecular Diagnostics
DNA Sequence Detection using Polymerase natural dna replication to rapidly amplify, or copy,
Chain Reaction (PCR) that sequence until there are thousands or millions
of copies. Because Pcr relies on amplification, it is
one of the essential methods underlying many
highly sensitive, meaning it can detect specific dna
molecular diagnostics is amplification, the process
segments that may be present at very low levels
of making copies of a specific dna or rna sequence
in the sample.
found in a sample (e.g., blood or tumor) until there
are so many copies that they can be detected and early researchers developed ways to detect the
measured. presence of Pcr-amplified dna by how far it traveled
in a gel in relation to other, different-sized, pieces of
there are a number of different amplification
dna when all the pieces were pulled by an electric
technologies, but polymerase chain reaction (Pcr)
current (gel electrophoresis). gel electrophoresis
testing is the most widely used and is considered a
can still be used, but now the use of fluorescent
work horse in molecular diagnostics. chemist dr. Kary
dyes, that bind to the target dna segment as it is
Mullis received a nobel Prize for inventing Pcr
amplified, enable automated instruments to monitor
in 1983.
the resulting fluorescence without using gels. in this
Pcr is a powerful tool for locating short segments of manner, technologies such as quantitative real-time
a gene where known critical mutations or variances Pcr not only can detect the presence of the target
can lead to altered cell functions associated with dna segment, but also quantify the amount of target
disease or altered function. Pcr tests for the presence dna that was originally present in the sample. this
of a portion of dna that has a known base sequence, can be used, for example, to determine the prevalence
employing the same enzymatic process used by of an infectious agent in the body.

Figure 4: Polymerase
chain reaction to amplify
Amplification Cycles dna by repeating
cycles that each duplicates
the base sequence in a
1 2 3 specific section of the
dna strand.

Purified DNA Primers choose DNA segment of interest, PCR amplifies

sample in cycles

The essenTials of DiagnosTics series: Molecular diagnostics 10

Molecular Diagnostics
Over time, the use of PCR evolved from diagnostics tube and checked to make sure that the DNA size is as
for single gene mutations, to amplification-based expected after the reaction. The amplified DNA
HIV viral load testing, to multiplex testing (testing for can then be analyzed in multiple ways depending on
multiple sequences at once), to high volume infectious the desired result. The DNA can be measured just
disease testing. Most PCR tests used chemical for size to check for large deletions or insertions of
reactions that rely on rapid cycles of temperature DNA, it can be measured for abundance, or it can
change to generate new strands of DNA. First, the be further prepped and sequenced to determine,
DNA must be extracted and purified from the raw nucleotide by nucleotide, what the DNA sequence in
human sample (e.g. blood, tissue, saliva, etc). This the sample is. In automated PCR systems, dNTPs can
is done using various scientific methods depending be tagged with dyes or other chemicals that can be
on the type of sample. For example, to extract DNA detected by the instruments as amplification occurs
from blood, the blood is separated to isolate the cells (“real-time PCR”), without requiring intervening
from surrounding fluids, is suspended in a solution to manipulation by a laboratory technician. The output
stabilize the cells and the cells are then broken open is generally an amplification curve.
with detergents. Additional chemicals are added to
Researchers continue to devote resources to
remove proteins and to keep the DNA from breaking
simplifying and decreasing the time for the PCR
apart. Finally, the DNA is separated from all of the
process. Some are working on decreasing the need
other products in a series of separation steps. Several
for the upfront DNA purification before the reaction,
life science companies have developed easy-to-use
and/or working on amplifying in less time. Finally,
kits for the purification of DNA that combine many of
newer isothermal methods that do not rely on
these steps, so it is not as complex and “hands-on”
temperature cycles have simplified testing equipment
as it was a decade ago.
and allowed versions to be produced for use outside
Then, to perform a PCR test, a researcher or the laboratory and even in quite elementary health
laboratorian will add multiple chemicals to a small care settings. Whereas PCR based diagnosis used to
tube or well in a plate. These chemicals include buffer, take days or weeks to perform by a trained molecular
a mix that contains the building blocks of the new biologist, some systems now can produce a patient
DNA strand called dNTP mix (dATP, dCTP, dGTP, result in as little as 30 minutes by non-laboratory
DTTP), salt (i.e., magnesium chloride or MgCl2), and personnel at the point of care.
primer mix. Primers are short pieces of DNA that
Companies have worked diligently to develop PCR and
have a complementary sequence to the particular
other amplification methods into the most powerful
target gene sequence of interest, which specifically
clinical diagnostic technologies to date. The transition
identifies the target, out of the whole DNA sample,
of these molecular diagnostics from highly specialized,
and ensures replication of that sequence of interest.
low volume laboratories to high volume core lab
Then, the purified DNA is added along with water, and
assays, and to point of care testing, has taken place
an enzyme called TAQ polymerase. TAQ is an enzyme
quite rapidly.
- a thermostable DNA polymerase - which drives the
amplification reaction by adding bases to the primer
to replicate the target sequence. The total volume in
the tube can be as little as 20 microliters, or less than
a drop of water. The tube or plate is sealed and placed
into a PCR thermocycler, which is an instrument that
rapidly heats and cools the amplification reaction to
mimic DNA replication much faster than it occurs
naturally. In non-automated PCR systems, once the
DNA is amplified, a small aliquot is taken from the

The Essentials of Diagnostics series: Molecular Diagnostics 11

Molecular Diagnostics
1985 1991 1998 2007+ 2011+
Kary Mullis Roche acquires First PCR-based >$1B clinical High-volume
invents PCR PCR from Cetus tests for HIV revenue from infectious
viral loads licenses and autmated PCR-
$0 clinical revenue available in labs products based platforms
begin to enter
core lab

1980s 1990s 2000s 2010s

note: adapted from Health advances aacc presentation by Kristin Pothier 2011
source: Washington g2 reports, Health advances interviews and analysis, roche company data.

Figure 5: adoption and impact of Pcr.

The Clinical Role

diagnostics are important decision making tools used in each of these stages. this section goes
for every stage of care – risk assessment, screening, on to discuss in more detail several examples of
diagnosis, staging and prognosis, therapy selection, how molecular diagnostics are making a critical
and monitoring. there are far too many molecular difference in patient care.
diagnostics in use to cover in this report, but Figure 6
provides an example of a molecular diagnostic being

Molecular Diagnostics Examples

There are hundreds of examples of molecular diagnostics across this continuum.
Risk Screening Diagnosis Staging and Therapy Monitoring
Assessment Prognosis Selection

Description Diagnostic Applied to Use for Assess Used to Recurrence

tests to high-risk definitive severity predict monitoring
complement patient to diagnosis and/or risk efficacy Monotoring
traditional identify and general of recurrence or safety for treatment
risk factors disease early cancer typing Inform response efficacy
adjuvent to specific
chemo treatments
decision

Molecular CF carrier MRSA TB OncoType Dx BRAF BCR-ABL

Diagnostic testing
HPV CT/NG MammaPrint KRAS HIV viral load
Example BRCA1
testing Her2

Figure 6: examples of molecular diagnostics across the continuum of care.

The essenTials of DiagnosTics series: Molecular diagnostics 12

 Molecular Diagnostics
Screening Test Example: HPV Screening/Diagnostic Test Example: Cystic Fibrosis
The fight against cervical cancer is largely a success The cystic fibrosis test is an example of a molecular
story in the United States and other developed diagnostic test that can be classified as a screening
countries, where the introduction in the 1940s of the test or a diagnostic test, depending on when it is used.
Papanicolaou (Pap) smear test to look for abnormal/ Cystic fibrosis (CF) is an inherited chronic disease
precancerous cervical cells has driven down death that mainly affects the lungs and digestive system and
rates from cervical cancer by as much as 70 percent. currently afflicts about 30,000 children and adults in
The CDC reports that cervical cancer affects about the United States (70,000 worldwide)4. About 1,000
12,000 women in the United States each year2, new cases are diagnosed each year.
however, and is the second leading cancer of women
CF is caused by an abnormality in the CF
worldwide, due to poorer access to health care in
transmembrane regulator (CFTR) gene that prevents
developing countries. Globally, cervical cancer affects
the normal movement of chloride ions across
about 500,000 women and kills about 250,0003.
membranes. The disease is characterized by a buildup
About 20 years ago, of mucus in the lungs, pancreas, and other organs of
researchers discovered the body. Over time, this results in patients having
The most a link between the serious breathing and digestive difficulties and an
successful treatment human papilloma average life expectancy that is less than 40 years.
virus (HPV) and
of cervical cancer cervical cancer. It
CF is seen in patients with two altered CFTR genes,
is delivered as a result of early one from each parent. Those that only have one copy
is now known that,
detection. The use of HPV tests of the altered gene are considered “carriers”, and
of the 100 or so
to identify women most likely while they do not have symptoms of CF, they can
genetically distinct
to develop cervical cancer has pass the gene to their children. There are about ten
types of HPV,
the potential to revolutionize million carriers in the United States. Therefore, “carrier
about fourteen are
treatment of this screening” of parents-to-be or pregnant women
associated with a high
disease. determines their CFTR gene status and helps families
risk of cervical cancer,
adequately prepare for the results. Testing is also
and just eight of those are
done on amniocentesis samples to directly assess
responsible for almost all cases.
CF status in the unborn child. Newborns are
Because HPV types are genetically identifiable, it has screened in all 50 states to assess CF status.
been possible to develop molecular diagnostic tests
One of the most common mutations causing CF is
to screen women for the high risk types. Identifying
the delta F508 deletion mutation which can be
women with these high risk types enables those
detected using a number of molecular techniques.
women to schedule more frequent exams and can
These tests use DNA amplification methods to
guide a physician to follow up more aggressively when
amplify key portions of the CFTR gene and look for
a Pap test yields abnormal or inconclusive results.
the mutations that cause CF.
In this way, molecular diagnostics are driving the
next advances in early detection and prevention of
cervical cancer after the historical success of the Pap
test. The American Cancer Society and other leading
professional societies recommend that women 21-65
years old should receive a Pap test (or other form of
cytological test) every three years, and women 30-65
years old should receive a cytological test together
with an HPV test every five years.
2
www.cdc.gov 3 www.path.org 4 www.cff.org

The Essentials of Diagnostics series: Molecular Diagnostics 13

Molecular Diagnostics
Normal Person CFTR Sequence Nucleotide: ATC ATC TTT GGT GTT
Amino acid: Ile Ile Phe Gly Val

506 508 510

Deleted in Delta F508

Person with Delta F508 Nucleotide: ATC ATC GGT GGT

Cystic Fibrosis CFTR Sequence Amino acid: Ile Ile Gly Val

506

Figure 7: Genetics in Cystic Fibrosis: A person with cystic fibrosis has a

deletion of three nucleotides in the CF gene which results in deletion of one
amino acid in the CF protein and the classic CF symptoms.

The American College of Obstetricians and allow physicians to quickly and definitively diagnose
Gynecologists (ACOG) and the American College CT/GC and prescribe antibiotics to the patient,
of Medical Genetics (ACMG) recommend offering clearing their infection and equally importantly,
testing for 23 common CFTR mutations to all women stopping spread of the diseases. A rapid (90 minute)
currently planning a pregnancy or seeking prenatal molecular CT/GC test for use at the point of care
care. This helps identify carriers and thereby informs has recently been developed, providing patients
their reproductive planning. Testing can also be done with an immediate diagnosis and helping to ensure
on the newborn or later in childhood to diagnose CF. appropriate follow up.
Diagnostic Test Example: Chlamydia/Gonorrhea Therapeutic Decision-Making Example: KRAS
One of the most common infectious disease tests An example of a molecular diagnostic that improves
is the molecular test used to detect chlamydia and therapeutic decision-making is K-Ras testing. K-Ras
gonorrhea (a CT/GC test). Because co-infection is a protein, encoded by the KRAS gene, that plays a
is common, a combined molecular test allows critical role in cell division, cell differentiation, and the
for amplification and detection of both Chlamydia self-destruction of cells. KRAS mutations that produce
trachomatis (CT) and Neisseria gonorrhoeae an abnormal, overactive K-Ras protein are found in
(GC) DNA. pancreatic, colorectal, lung and other cancers.
Chlamydia is the most frequently reported bacterial Detecting a KRAS mutation is used to determine
sexually transmitted infection in the US, with over 1.4 patient suitability for certain therapies in colorectal
million diagnosed and an additional 1.4 million people and lung cancer. Anti-EGFR (epidermal growth
with the disease who are not diagnosed. Gonorrhea factor receptor) drugs (Erbitux (cetuximab) and
is also common, with an estimated 700,000 with the Vectibix (panitumumab)) are key therapies for those
disease but less than half actually reported5. cancers, but patients whose cancers contain KRAS
typically fail to respond to these anti-EGFR therapies.
These sexually transmitted diseases are caused
Therefore, Erbitux and Vectibix labels recommend
by bacteria that can be detected through urethral
against prescribing the drugs for patients with certain
or cervical samples (as well as a urine sample for
KRAS mutations. KRAS mutation testing is typically
gonorrhea) using molecular testing. These tests
performed for patients with metastatic colorectal
5
www.cdc.gov

The Essentials of Diagnostics series: Molecular Diagnostics 14

Molecular Diagnostics
cancer or metastatic lung cancer via direct DNA The Future of Molecular Diagnostics
sequencing or PCR and other amplification-based
This report includes only a few of the hundreds of
methods.
examples in which the latest molecular technology is
KRAS mutation testing is just one of a growing being used for rapid diagnosis and to aid physicians in
number of examples of molecular diagnostics that improving clinical outcomes for their patients. Since
help inform a physician’s decision making regarding we are only in the early stages of understanding the
appropriate treatment. Identifying which patients complex fields of genomic and proteomics, however,
are unlikely to respond to a specific kind of therapy and diagnostic technologies are evolving rapidly,
reduces the costs of ineffective treatment (measured it seems fair to say that much of the promise of
in dollars, unnecessary side effects, and time that molecular diagnostics is still ahead of us.
could be spent on other treatments).
As we understand more about the complexity
Therapeutic Monitoring Example: HIV of diseases, there are an increasing number of
molecular biomarkers that must be identified to
Finally, many molecular diagnostics improve
fully characterize the many subsets of populations
monitoring and management of disease. Some of the
that have highly variable symptoms and clinical
most common monitoring molecular tests are tests for
courses. New technologies are making it possible to
HIV viral load in patients affected with HIV. HIV viral
gather more information to more fully characterize
load tests are amplification-based tests that measure
the disease state, the best treatment alternatives
the amount of HIV RNA to determine how many
for a patient, or in the case of infectious disease,
copies of the virus are present (measured in copies
rapidly identify a specific pathogen out of many that
per milliliter of blood). Keeping viral levels as low as
could be responsible for a particular infection (e.g.,
possible is key to staying healthy with HIV.
respiratory viruses). Approaches that may address
Because researchers and physicians have made great this need for increasing amounts of data include
strides in the care of HIV patients in the three decades multi-marker or multiplexed approaches (methods
since HIV was first detected, essentially transitioning that can test for multiple biomarkers in a single assay)
it into a chronic disease in many developed countries, and gene sequencing that can detect multiple defects
the HIV viral load test has become an essential tool as associated with a particular subset of patients with a
patients cycle through different antiretroviral therapies disease. Practical application of these methods can be
and develop resistance to certain therapies. Periodic challenging, due to complexity of the assays involved,
HIV viral load tests indicate whether or not the but these challenges are being met by integration
disease is progressing, either because an untreated of molecular methods with advanced information
patient’s immune system can no longer suppress technology (IT) and data analytic methods.
replication of the virus, or because the therapy for
New technologies also will bring greater efficiency
a patient in treatment is no longer working.
to current methods. There has been a great deal of
The HIV viral load test is not used for initial diagnosis excitement about the promise of nanotechnology to
(this is often done with antibody tests), but typically support the development of the molecular diagnostics
also is used upon initial diagnosis as a staging field. Companies have commercialized technology
diagnostic, to get a first report on the progression based on gold nanoparticles that allow increased
of the disease prior to treatment. sensitivity and specificity for molecular diagnostics
assays and that also enable multiplexing of assays.
Other nanotechnology-enabled approaches include
innovative ways to sequence DNA in which a single
DNA strand is extruded through a nanopore and
the sequence is “read” base pair by base pair.

The Essentials of Diagnostics series: Molecular Diagnostics 15

Molecular Diagnostics
Finally, molecular diagnostics at the point of care is • The ability and capacity to capture, store and
making great strides. Advancements have focused analyze the tremendous and rapidly increasing
on ease of use, producing tests that can be performed amount of diagnostics data, particularly with gene
in less specialized laboratories, rapid turn-around sequencing tests, is an ongoing challenge. IT
time, and appropriately small size and simplicity for systems must have enough power to process
resource-constrained settings. and analyze these huge volumes of information. In
addition, another key challenge is the need for data
Molecular diagnostics are truly transformative
standards that would speed interpretation of these
technologies. Yet excitement about the future of
complex genomic data sets and make the results
this field must be tempered by recognition of the
relevant for specific patients. This task is further
challenges of continuing innovation and timely patient
complicated by the fact that the patient’s medical
access. The challenges include regulatory hurdles,
record is often in many unconnected places,
coverage and reimbursement issues, and practical
whether in multiple out-patient offices, multiple
and ethical (privacy) issues associated with generating
hospitals, or multiple pharmacy systems.
and storing large volumes of highly detailed and
personal health information. • Finally, ethical questions are also being raised
about the disclosure and use of genetic
• The fast pace of technological development in
information. Genetic markers are being identified
molecular diagnostics, and the rapidly growing
that can signal the propensity of a patient to
body of research regarding biomarkers,
develop a disease over their lifetime and the
challenge regulatory bodies to stay current.
question has been raised of how, if at all, such
Clear understanding of regulatory expectations
information should be disclosed, particularly in
for multiplex tests, companion diagnostics and
cases where prevention or treatments are not
next generation sequencing as well as the
yet available. Policy makers continue to grapple
increasing investments necessary to generate
with these challenges.
the clinical evidence required for approval or
clearance will be integral.
• There are questions regarding the patenting of
gene sequences currently working their way
through the courts. The ability
• Insurance coverage and payment for diagnostics to interconnect
is under constant downward pressure as payers try all lab test results for a patient
to keep down escalating health care costs. Despite
at diagnosis and over time, no
the strong value proposition of diagnostics overall,
payers are increasingly asking for health economic matter what type of test or where
evidence supporting the use of specific tests, which it was performed, is one of the
often requires further evidence generation above most important next steps to take
and beyond regulatory requirements.
in delivering the most informed,
economical and holistic care
to the patient.

The Essentials of Diagnostics series: Molecular Diagnostics 16

The Future of Molecular Diagnostics
Conclusion

This report has only touched the surface of molecular AdvaMedDx

diagnostics today. The power of the science, clear links The world’s leading diagnostics manufacturers
to clinical outcomes, and the technological advances established AdvaMedDx to advocate for the power of
that are allowing molecular diagnostics to go from medical diagnostic tests to promote wellness, improve
a translational research tool to mainstream clinical patient outcomes, and advance public health in the
applications are impressive. We also are witnessing United States and abroad.
the transition of these technologies to settings outside
the lab, where their impact may be even greater. AdvaMedDx represents one of the most dynamic
and innovative sectors in the health care system.
DxInsights and AdvaMedDx will publish additional Diagnostics influence as much as 70% of health
reports that will continue to tell this story. These care decision making. These tests—performed in
reports will include exploration of next generation laboratories, at the hospital bedside, in doctor’s
sequencing technologies, health IT and treatment offices, in medical clinics, and in the home—facilitate
of complex algorithms in diagnostics, companion evidence-based medicine, improve quality of care,
diagnostics, and global diagnostics needs. promote wellness, enable early detection of disease
and often reduce health care costs.
Acknowledgments
For more information, news, and educational materials
Introduction to Molecular Diagnostics: relating to this report or other diagnostic topics, please
The Essentials of Diagnostics Series visit our web sites.
Copyright ©2013 DxInsights / AdvaMedDx www.dxinsights.org

DxInsights is a non-profit organization formed to advameddx.org

elevate the understanding of diagnostics in the This report was written by the DxInsights and
face of rising health care costs. Created by industry AdvaMedDx teams, guided by Kristin Pothier and
veterans involved in all facets of diagnostics including Ray Woosley, DxInsights, Andrew Fish and Tharini
development, commercialization, regulatory, Sathiamoorthy, AdvaMedDx, and a team of
reimbursement and communications, DxInsights is industry and institutional reviewers.
focused on expanding the role diagnostics play in the
future of health care and personalized medicine.
The goal of DxInsights is to conduct educational
programs to guide industry colleagues, policy
makers, payers and health care advocates to greater
appreciation of the power and value of diagnostics
and their direct impact on improving patient
outcomes and reducing costs.

The Essentials of Diagnostics series: The Future of Molecular Diagnostics 17

Glossary of Common Molecular
Diagnostic Terms
Allele Gene
An allele is one of two or more forms of the DNA A hereditary unit consisting of a sequence of DNA
sequence of a particular gene. Different DNA that occupies a specific location on a chromosome
sequences (alleles) can result in different traits, such and determines a particular characteristic in an
as color of skin, hair or eyes. If both alleles are the organism. Genes undergo mutation when their DNA
same, the person is termed a homozygote. If the sequence changes during cell division.
alleles are different, the person is a heterozygote.
In Vitro Diagnostics (IVD)
Amplification In vitro diagnostic products are those reagents,
Amplification of DNA is a process in which the instruments, and systems intended for use in
polymerase chain reaction (PCR) is repeated for a diagnosis of disease or other conditions, including a
number of cycles or rapidly changing temperatures determination of the state of health, in order to cure,
to exponentially increase the number of copies of mitigate, treat, or prevent disease or its sequelae.
a specific target region of a gene. Such products are intended for use in the collection,
preparation, and examination of specimens taken
Biomarker
from the human body.
A biological property or substance(s) that is a sign
of a normal or abnormal process, or of a condition Microarray
or disease. A biomarker is used to determine how A microarray is a multiplex lab-on-a-chip. It is a 2D
patients respond to treatments. array on a solid substrate (usually a glass slide or
silicon thin-film cell) that assays large amounts of
Diagnostics
biological material using high-throughput screening
The use of clinical tests to inform clinical decision
methods.
making. The area includes both tests conducted on
specimens from the body (i.e., in vitro diagnostics) Molecular Diagnostics
and imaging tests (e.g., in vivo diagnostics), for the Diagnostic tests that identify a disease, predisposition
purpose of disease prediction, screening, diagnosis, for a disease, or progress in treating a disease by
treatment selection, prognosis and monitoring. detecting specific molecules such as DNA, antibodies,
and proteins.
DNA
DNA is the acronym for deoxyribonucleic acid, the Mutation
code used within cells to form proteins. In cells, In molecular biology and genetics, mutations are
DNA usually exists as two long intertwined chains changes in a genomic sequence: the DNA sequence
twisted into a double helix and joined by hydrogen of a cell’s genome or the DNA or RNA sequence of
bonds between the complementary bases adenine a virus. Mutations are caused by radiation, viruses
and thymine or cytosine and guanine. The chains are and mutagenic chemicals, as well as errors that
composed on nucleotides (a combination of any one occur during DNA replication. Mutation can result in
of the nucleic acid sequence of nucleotides in DNA several different types of change in DNA sequences;
determines individual hereditary characteristics. these can have either no effect, alter the product
of a gene, or prevent the gene from functioning
Esoteric Test
properly or completely. Due to the damaging effects
The analysis of ‘rare’ substances or molecules that are
that mutations can have on genes, organisms
not performed in a routine clinical lab.
have mechanisms such as DNA repair to remove
mutations.

The Essentials of Diagnostics series: Glossary of Common Molecular Diagnostic Terms 18

Glossary of Common Molecular
Diagnostic Terms
Nucleic Acids RNA
Nucleic acids are any of various complex organic acids RNA is an acronym for ribonucleic acid. RNA is chain
(e.g., DNA or RNA) that are composed of nucleotide of alternating phosphate and ribose units with the
chains. bases adenine, guanine, cytosine, and uracil bonded to
the ribose. RNA molecules are usually single stranded
Nucleosides
and involved in protein synthesis.
Any of a class of organic compounds, including
structural subunits of nucleic acids. Each consists Sequencing
of a molecule of a five-carbon sugar (ribose in RNA, The technique used to map out the sequence of the
deoxyribose in DNA) and a nitrogen-containing nucleotides that comprise a strand of DNA.
base, either a purine or a pyrimidine. The base uracil
Thermocycler
occurs in RNA, thymine in DNA, and adenine,
is a laboratory instrument used to amplify segments of
guanine, and cytosine in both, as part of the
DNA using PCR. The instrument has a thermal block
nucleosides uridine, deoxythymidine, adenosine
which holds amplification tubes for each reaction. The
ordeoxyadenosine, guanosine or deoxyguanosine,
thermocycler raises and lowers the temperature of the
and cytidine or deoxycytidine. Nucleosides
block in programmed steps to perform the reaction.
usually have a phosphate group attached,
forming nucleotides.
Nucleotides
Nucleotides are any of several compounds that consist
of a ribose or deoxyribose sugar joined to a purine
or pyrimidine base (adenosine, cytosine, thymidine,
guanine or uracil) and to a phosphate group and that
are the basic structural units of nucleic acids (as RNA
and DNA).
Polymerase Chain Reaction (PCR)
A technique for amplifying DNA sequences in vitro by
separating the DNA into two strands and incubating
it with oligonucleotide primers and DNA polymerase.
It can amplify a specific sequence of DNA by as many
as one billion times and is important in biotechnology,
forensics, medicine, and genetic research.
Polymorphism
The existence of a gene in several allelic forms
of >1% frequency; also: a variation in a specific
DNA sequence.
Primer
A segment of DNA or RNA that is complementary to
a given DNA sequence and that is needed to initiate
replication by DNA polymerase.

The Essentials of Diagnostics series: Glossary of Common Molecular Diagnostic Terms 19