Larry H Bernstein, MD, FCAP
Principal & CSO, Triplex Medical
Chief Scientific Officer
Leaders in Pharmaceutical Business
Intelligence, Trumbull, CT
The Future of the CLS Profession Over the Next Decade
• Point # 1 Pathology and CLS professions have
changed every decade
• Point #2 Pathology and CLS professions are
changing today
Outline
Prime Movers of Change
• Human Genome Project post decade
projected forward
• Biotechnology boom
• Information technology
Breakthroughs
• Driven by Personalized Medicine
• Genomic and Allied –OMICs- technology
• Communication Science and Open and
Interactive Architectures
• Mathematical Innovation in Classification and
Complexity
• Nanotechnology
• Integrative Scientific Discovery
What of Metabolomics and Metabolic Profiling?
• Metabolomics is the measurement of small molecules
that interact with membrane receptors
• involved with regulation of genomic transcription and
cellular regulation
• involved with upregulation or downregulation of
metabolic processes
• provide targets for disease treatments
• provide further “analytes for diagnosis
• prediction of short-term or long-term outcomes.
Impact on CLS
• Metabolomics is extension of “regulatory
activity of genome”, formerly deemed
“DARK MATTER”
• Metabolomics involves the interaction with
the environment (food, stress)
• Metabolomics takes into consideration
“mutational risk” and health or disease
Expanded Role of Laboratory
• Improved tie in with provision of information to not
only the healthcare workers, but also the patient.
• Achieve turnaround times for critical results through
better workflow
• Greater control and access to a next generation of
point-of-care technology integrated with the laboratory
database, and a restructured electronic health record.
• Despite the hype about the BIG DATA revolution, this is
achievable in the system here proposed because there
is a published model to achieve this
George Lundberg, MD, MedPage Today
• The alphabet soup that describes cancer
genes, mutations, and pathways includes
• EGFR, KRAS, EML4-ALK, AKT, BCL, MITF, CDK,
C-KIT, GNAQ, BRAF, and GNA11.
• pathologists may become clinical molecular (&
diagnostic) specialists – determining:
• what molecular (and other) tests, if any,
should be done
Methods Used
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Microarrays and microfabrication
Separations technology
High throughput and
Multiple analytes
Disease Identification and Risk Management
• Carbohydrate metabolism - insulin, C-peptide
• Lipid Metabolism – HDL, LDL, small dense LDL
particles, Apo E
• Proteins – proteomics, immunoglobulins,
coagulation, CRP (inflammatory), IL-1, 6, 8..
• Cellular regulation – PPARγ, p53, HER2, etc.
• Cytology, hemocytometry, flow cytometric
analysis
Digital Microscopy
• 3-D visualization
• Receptor staining
• Fresh Tissue
• Comment: Radiographic imaging
Mathematical Analysis
Comparison of LDA vs. Logit Analysis
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Under LDA assumptions
the discriminant function is linear in X
predicted probability of Z=1 follows logistic
distribution
coefficient estimates more efficient than
logistic regression approach
Problem: distributions of covariates not
gaussian
Realtime Clinical Expert Support and Validation System
• a software system that is the equivalent of an
intelligent Electronic Health Records Dashboard
• provides empirical medical reference and
suggests quantitative diagnostics options
• gathers medical information
• generates metrics
• analyzes data in realtime
• provides a differential diagnosis
Given medical information of a patient
• the system builds its unique characterization
• provides a list of other patients that share this
unique profile
• utilizing the vast aggregated knowledge
(diagnosis, analysis, treatment, etc.) of the
medical community
patient profiling
• accurate patient profiling and inference
methodologies
• anomalous subprofiles are extracted
• and compared to potentially relevant cases
Sample Report
Patient Course Summary
Insights into the chemical structure of DNA
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the basic building blocks of DNA and proteins,
of nucleotide and protein-protein interactions,
protein folding, allostericity,
genomic structure,
DNA replication,
nuclear polyribosome interaction, and
metabolic control.
Flow of genetic information
Genomic Methods
Novel Adipocyte Secreted Protein in
Liver Glucose Regulation
• A particular type of protein (hormone) found
in fat cells helps regulate how glucose (blood
sugar) is controlled and metabolized (used for
energy) in the liver.
• Switching off this protein leads to better
control of glucose production from the liver,
revealing a potential new target that may be
used to treat type 2 diabetes and other
metabolic diseases.
ProsVue PSA
NZDiA ProsVue Timeline
Mechanisms of Acute Coronary Syndromes and Their
Implications for Therapy
Peter Libby, M.D.
N Engl J Med 2013; 368:2004-2013
The notion that heart attacks develop from
coronary-artery stenosis is an oversimplification
of a process involving
lipid metabolism,
inflammation,
macrophage activation,
collagen breakdown, and
…….plaque rupture……(not always)
NO Endothelial Relaxation
Nitric Oxide cycle and vascular
relaxation/vascular stiffness
Genome Wide Association studies (GWAS)
• Genome-wide association studies (GWAS) aim to
identify causative genes for common disease
such as diabetes, obesity and stroke by
• analysing single nucleotide polymorphisms
(SNPs).
• SNPs are identified that show association with a
disease,
• they are unlikely to be the actual genetic variant
involved in causation of that disease
• most individual SNPs for common diseases raise
risk by about only 20-40%
Genetic Links in Common Disease
• variants in the same gene confirmed link
between APOC3 and early-onset heart attack
• APOC3, involved in fat metabolism, may delay the
catabolism of triglyceride-rich particles
• Polymorphisms in the human APOC3 gene and
promoter have been associated with
• • lipoprotein profile
• • cardiovascular health
• • insulin (INS) sensitivity
novel polymorphism of the CYP2J2 gene
• coronary artery disease in Uygur population in
China
• Cytochrome P450 (CYP) 2J2 is expressed in the
vascular endothelium and
• metabolizes arachidonic acid to biologically
active epoxyeicosatrienoic acids (EETs)
genomics in discovery of therapeutic targets
• key drivers of cellular proliferation,
• stepwise mutational changes coinciding with
cancer progression
• potential therapeutic targets for reversal of
the process
Melanoma Drugs approved by FDA
• a B-Raf inhibitor aimed to treat melanoma
patients harboring V600E mutation
• a MEK inhibitor that was shown in phase III
clinical trials to be efficient for treating
melanoma patients with BRAF V600E or V600K
mutations
• These new two drugs are now joining the first
two drugs approved in 2011 to treat metastatic
melanoma that are already in clinical use
Companion Diagnostics Testing
• The introduction of the two drugs was co-approved in
concert with the THxID BRAF test from BioMérieux.
• This PCR-based BRAF test is designed to determine
whether a melanoma patient harbors the V600E or
V600K BRAF gene mutation and will assist directing the
correct treatment to be given to patients.
• this companion diagnostic approved for BRAF
mutation detection follows the approval of Roche’s
cobas 4800 BRAF V600 Mutation Test in August 2011
• the association of diagnostics with treatments is a step
further by pharmaceutical and diagnostic companies
toward establishing personalized medicine
New test gives better stratification for
prognosis of CLL
• Innovation Department - Hospital Clinic
Barcelona
• classify CLLs into 3 new prognostic groups:
• • MBC-like
• • intermediate
• • NBC-like
• using only 5 epigenetic biomarkers
epigenetic signature has independent
prognostic impact
• used bisulfite pyrosequencing primers designed
to detect methylation of the 5 biomarkers.
• There are four possible formats to develop this
test:
• • Pyrosequencing
• • PCR + restriction enzymes
• • Hybridization
• • Immunoprecipitation + qPCR
Rationale of Innovation
current classification of M-CLL (mutated) and UCLL (unmutated) is not used in clinical protocols
to decide upon patient treatment and follow-up
After 10 years only
Group
Need Treatment (%)
MBC-like
20
Intermediate
43
NBC-like
100
This test will allow
• - Improve the quality of life of CLL patients
• - Improve management strategies that are
more adjusted to the biology of this disease
• - Reduce the health care costs
Epigenome
The suite of epigenetic modifications, collectively forming a dynamic
epigenetic code, has long been known to operate as the manager of
the genomic blueprint,
wielding cell-dependent control over gene expression at discrete times
throughout development.
Our understanding of the disease epigenome has expanded, with the
inclusion of numerous gene mutations and epimutations involved in
producing aberrant gene expression.
these findings form inextricable causal links between our underlying
genome,
a dynamic epigenome, and
the functional (disease) consequences stemming from both.
Non-Invasive Test for Early Detection
of Colorectal Tumors
Dr. Guro Elisabeth Lind and a team from Oslo
University Hospital have identified altogether 12
epigenetic biomarkers for early detection of
colorectal cancer
a six-gene panel could outperform several
previously published epi-markers, including VIM
and SEPT9 which are included in commercial
tests for early detection of colorectal cancer
using stool and blood, respectively
a disease defined primarily by its
genetic fingerprint
(NY Times) By GINA KOLATA Published: May 1,
2013
Jeff Boyd, Fox Chase
the similarity among breast, ovarian and
endometrial tumors was the best example yet of
the idea that cancers are more usefully classified
by their gene mutations than by where they
originate.
CD47 in cancer immunity
• Evasion of immune recognition is a major
mechanism by which cancers establish and
propagate disease.
• Recent data has demonstrated that the innate
immune system plays a key role in modulating
tumor phagocytosis through the CD47-SIRPα
pathway.
• Careful development of reagents that can block
the CD47/SIRPα interaction may indeed be useful
to treat many forms of cancer
Akt in cancer
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Mechanisms for Akt pathway activation include
loss of tumor suppressor PTEN function,
amplification or mutation of PI3K,
amplification or mutation of Akt,
activation of growth factor receptors,
inactivation of the translation repressor protein
4E-BP1, and
• exposure to carcinogens.
• Akt knockout mice demonstrate that Akt is
required for both cancer cell survival and
oncogenic transformation.
• That activation of Akt is oncogenic, could be
explained by
preventing normal apoptosis of cells, thereby
enabling accumulation of more oncogenic
mutations in these cells.
Akt controls many cancer related cellular
processes such as
• cell metabolism,
• growth and survival,
• proliferation, and
• motility,
all of which contribute to tumor initiation and
progression.
What are lncRNAs?
• Non-coding RNA is now known to make up the
majority of transcribed RNAs and in addition
to those that carry out well-known
housekeeping functions (e.g. tRNA, rRNA etc),
many different types of regulatory RNAs have
been and continue to be discovered.
• Many of these non-coding RNAs are thought
to have a wide range of functions in cellular
and developmental processes.
• This large and diverse class of transcribed RNA
molecules with a length of more than 200
nucleotides do not encode proteins.
• Their expression is developmentally regulated
and lncRNAs can be tissue- and cell-type specific.
• A significant proportion of lncRNAs are located
exclusively in the nucleus.
• They are comprised of many types of transcripts
that can structurally resemble mRNAs, and
• are sometimes transcribed as whole or partial
antisense transcripts to coding genes.
Types of molecular mechanisms that
may be involved in lncRNA function
• The Signal archetype: functions as a molecular
signal or indicator of transcriptional activity.
• The Decoy archetype: binds to and titrates away
other regulatory RNAs or proteins.
• The Guide archetype: directs the localization of
ribonucleoprotein complexes to specific targets.
• The Scaffold archetype: has a structural role as
platform upon which relevant molecular
components (proteins and or RNA) can be
assembled.
Similarities and differences
between mRNA and lncRNA
mRNA
lncRNA
Tissue-specific expression
Tissue-specific expression
Form secondary structure
Form secondary structure
Undergo post-transcriptional processing,
i.e. 5’cap, polyadenylation, splicing
Undergo post-transcriptional processing,
i.e. 5’cap, polyadenylation, splicing
Important roles in diseases and
development
Important roles in diseases and
development
Protein coding transcript
Non-protein coding, regulatory functions
Well conserved between species
Poorly conserved between species
Present in both nucleus and cytoplasm
Predominantly in nucleus
Total 20-24,000 mRNAs
Predicted 3-100 fold of mRNA in number
Expression level: low to high
Expression level: very low to moderate
Structural DNA nanotechnology
[1] Hybridization
The self-association (self=assembly) of complementary nucleic acid molecules or
parts of molecules, is implicit in all aspects of structural DNA nanotechnology
[2] Stably branched DNA
the combination of in vitro hybridization and synthetic branched DNA that leads to the
ability to use DNA as a construction material
[3] Convenient synthesis of designed sequences.
Biologically derived branched DNA molecules, such as Holliday junctions, are
inherently unstable, because
they exhibit sequence symmetry; i.e., the four strands actually consist of two pairs of
strands with the same sequence.
Origami
DNA origami utilizes the genome from a virus
together with a large number of shorter DNA
strands to enable the creation of numerous
DNA-based structures (Figure 1)
The shorter DNA strands forces the long viral
DNA to fold into a pattern that is defined by the
interaction between the long and the short DNA
strands
DNA nanotechnology and Biological Application
Drug delivery
Biosensors
capable of picking up very specific
biological signals and converting them into
electrical outputs that can be analyzed for
identification.
Computing
Reinventing as a way of life
Daniel Burrus, author of “Flash Foresight”
wrote: “Today you cannot just reinvent now and
then: to survive and thrive in a time of vertical
change, you have to be redefining and
reinventing yourself continuously. --- The
organizations that are winning in the new
century don’t bother competing; they leapfrog
the competition by redefining anything and
everything about their business.”
The ultrafiltration alternative for
Hormones & Therapeutic Drugs
Imagine an automated workstation dedicated to rapidly
prepare plasma ultrafiltrates.
This workstation does not require centrifugation, and the
same vessel used to prepare the ultrafiltrate, will be
presented to an immunoanalyzer with a regular menu of
immunoassays for measuring “Total” hormones and
drugs.
The ultrafiltrate will allow us to measure “Free”
hormones and drugs.
The workstation will be integrated in an automated line,
and a standalone workstation will be available for labs
that are not automated.
Drug Failures
• Drug failure rates, the cost to develop drugs
and healthcare costs are at an all-time high.
• These facts are what are driving the new
paradigm in drug development.
Companion Diagnostics
The value companion diagnostics bring to pharma lies in their ability to mitigate risk.
They can be the difference between drug approval and failure.
Increasingly, pharma companies are integrating companion diagnostic programs
and are developing them in conjunction with their drug development programs.
Companion diagnostics can provide an extra layer of protection and
a way for pharma companies to get efficacious drugs on the market
without presenting safety risks to discrete patient cohorts.