1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making 2. Identify the utility of these diagnostic approaches with some examples 3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings

1. Utility of Molecular Diagnostics in
clinical decision making
Nikhil Phadke, PhD
GenePath Dx, Pune, India
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2. This presentation is the property of
Learning Objectives
1. Be familiar with next generation molecular diagnostic
techniques that can provide guidance in clinical
decision making
2. Identify the utility of these diagnostic approaches
with some examples
3. Be aware of the challenges that exist in
implementing these tools as part of the routine
clinical decision making process, especially in
resource limited settings

3. This presentation is the property of
The Basics

4. This presentation is the property of
mt
16.6 Kb
37
1013 cells
Our DNA
Haploid Human Genome
~ 3.09 * 109 bp
Distributed in 23
chromosomes & mitochondria
Coding regions ~ 2%
(85% of all known mutations)
Total Genes 25,000 - 45,000
(Histone 500bp – Dystrophin 2.2 Mbp)

5. This presentation is the property of
Where can things go wrong?
What do we need to detect?
Analogy (using plain English)
♀THE DOG BIT THE CAT | THE CAT ATE THE RAT
♂THE DOG BIT THE CAT | THE CAT ATE THE RAT
Presence and quantification of foreign nucleic acids (e.g.
infectious agents like viruses and bacteria)
COW EGG PIG ASS
Single nucleotide polymorphisms (SNPs) (e.g. Sickle Cell
Anemia)
THA DOG BIT THE CAT
THE DOG BIT THE BAT
Insertions / Deletions (InDels) & Nucleotide Repeat
Polymorphisms – can cause frameshifts (e.g. Fragile X,
Huntingtons Disease)
THE EDO GBI TTH ECA T
THD OGB ITT HEC AT
Copy number variations (CNVs) e.g. (Deletion,
Duplications such as Her2 amplification in some Breast
Cancers)
THE DOG BIT THE CAT THE DOG BIT THE CAT
THE DOG DOG BIT THE CAT
OR
THE BIT THE CAT
Inversions (e.g. Factor VIII Haemophilia) THE GOD BIT THE CAT
TAC EHT TIB GOD EHT
Translocations (e.g. chronic myelogenous leukemia - CML
Philadelphia chromosome 22 & 9 translocation causes
BCR-ABL fusion)
THE DOG BIT THE CAT ATE THE RAT
THE CAT ATE THE DOG BIT
Loss of Heterozygosity (LoH) (e.g. retinoblatoma) /
Uniparental Disomy (e.g. Prader Willi Syndrome)
THE DOG BIT THE CAT
THE DOG BIT THE CAT
THE DOG BIT THE CAT

6. How do we detect these anomalies?
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Analysis scope
Single/ Few
Defined
Variations
Multiple/
Unknown
Variations
Cytogenetics Molecular Genetics
FISH
Karyotyping
(& Spectral)
(digital) PCR
SNaPshot
Sanger
Sequencing
MLPA
dHPLC/ HRM
Microarrays
Next Generation
Sequencing
Low resolution High resolution
M-FISH

7. The evolution of sequencing technologies
1970s 80s and 90s 2000s 2010s
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8. Traditional Capillary/Sanger Sequencing
Chromatogram
+ -
Shorter Longer
fragments fragments
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Nucleotides dA dT dG dC,
ddA ddT ddG ddC
Polymerase
Sequence X-X-X-T-T-G-S-T-A-T
Complementary Template Y-Y-Y-A-A-C-W-A-T-A
Primer X-X-X
X-X-X-T
Synthesized X-X-X-T-T
labeled DNA X-X-X-T-T-G
fragments X-X-X-T-T-G-C
X-X-X-T-T-G-G
X-X-X-T-T-G-C-T
X-X-X-T-T-G-C-T-A
X-X-X-T-T-G-C-T-A-T
X-X-X-T
X-X-X-T-T
X-X-X-T-T-G
X-X-X-T-T-G-C
X-X-X-T-T-G-G
X-X-X-
T-T-G-C-T
X-X-X-T-T-G-C-
T-A
X-X-X-
T-T-G-C-T-A-T
Capillary Electrophoretic
Separation
Sequencing Reaction

9. Pros and Cons of Capillary Sequencing
- Slow, limited throughput
- Expensive (per base)
- Limited sensitivity
- Not ideal for multiplexing
- Not ideal for multigenic or
large gene analyses
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 Gold standard
 Relatively long reads (700 -
1000 nucleotides)
 Nobel Prize for Fred Sanger
in 1980
 Used for Human Genome
Project (3000 scientists, multiple
centers, 6 countries, over 13
years, cost US$ ~ 3B)

10. Next (2nd) generation sequencing (NGS)
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Library Preparation
Data Capture &
Data Analysis
Fragmentation /
Targeted Amplification
Addition of Priming &
Multiplexing adapters
Massively
Parallel Sequencing
by Synthesis
Clonal amplification
(Emulsion/Bridge PCR)

11. Single molecule (3rd gen) sequencing
Single Molecule Real Time Nanopore Sequencing
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Sequencing
dA dT
dG dC
Zero-mode
Waveguide
Labeled
nucleotides
Anchored
Polymerase
Detection of incorporated
polymerase at base of ZMV
DNA
unwound,
ratcheted
through
protein
graphene
nanopore
Nucleotide detected
based on conductivity

12. Clinical applications of NGS
• Sequencing: de novo genome, resequencing, exome,
targeted, transcriptome profiling, metagenome analysis
• Large genes / multiple genes / multiple samples
• Ultra-deep sequencing for rare mutations and circulating
nucleic acids (e.g. plasma tumor DNA and maternal DNA for
non-invasive prenatal diagnosis)
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13. Digital PCR – An evolution of PCR
Conventional PCR Real-time PCR Digital PCR
Real time detection in
closed system
High Low
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Temp
Thermal cycling
Post PCR gel
electrophoretic analysis
Fluorescence
Cycles
Negative
End-point detection
of droplets
+ -
+
-
+
+
+
+
+
+
-
+
dA dT dG dC Polymerase + Primers dA dT dG dC
+ (Probes) +
Nucleotides + Target;
Individual Target DNA
physically separated
Polymerase + Primers
+ (Probes) +
Nucleotides + Target
in a
Homogenous system

14. Some Examples
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15. Molecular diagnostics across medical disciplines
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Pathogen
identification
e.g. MTB
Pathogen
quantification
e.g. HIV load
Drug
resistance
status e.g.
MRSA
Predictive testing
e.g. BRCA1/2
Companion
diagnostics e.g.
EGFR, HER2
Disease
monitoring e.g.
BCR-ABL
Prognostic testing
e.g. ‘OncotypeDx’
e.g. Neonatal
Diabetes,
Congenital
Adrenal
Hyperplasia and
Prader Willi
Syndrome
Transplantation
e.g. HLA typing
and chimerism
monitoring
Blood disorders
e.g.
Thalassemia
and DVT
e.g. Trisomy 21
testing by
amniocentesis,
CVS, or NIPD
Infectious
Diseases
Endocrinology
Hematology
Identity
Prenatal
Testing
Oncology

16. The utility of BRCA testing
1. Modify surveillance options
2. Suggest risk reduction measures
3. Provide treatment guidance
4. Understand risk to other family members
5. Potential for pre-implantation diagnostics (PIGD)
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17. BRCA Testing in India in 2012 (Sanger Seq)
58 year old female diagnosed with Breast Cancer with
family history of HBOC; common Indian mutations
tested
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BRCA1 Exon 2 185del AG
BRCA1 Exon 8 632 dupT
BRCA1 Exon 12 IVS17+1, G>A, 5154delC
BRCA2 Exon 9 IVS8-2, A>G(c.682-2 A>G)
BRCA2 Exon 25 W3127X
All negative. Results inconclusive

18. BRCA Testing in India in 2014 (NGS Panel)
High penetrance breast cancer susceptibility genes
BRCA1, BRCA2, TP53, PTEN, STK11, CDH1
Moderate susceptibility breast cancer genes:
ATM, CHEK2, PALB2, BRIP1, NBN, RAD51C, RAD51D
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Other genes in the panel:
MEN1, CDC73, DICER1, MUTYH, NF2, MSH2, BMPR1A, APC, KIT, RET, SMAD4,
RB1, VHL, EPCAM, ALK, CDK4, AIP, MSH6, MET, MLH1, SDHAF2, SDHB, SDHD,
SDHC, SUFU, WT1, TSC2, TSC1, PMS2.
Pathogenic frameshift BRCA1 Mutation Detected c.933_933delT

19. Genetic testing in Neonatal Diabetes
• ATP-sensitive potassium channel (KATP) consists of two
subunits - SUR1 (ABCC8 – 4000bp) and Kir6.2 (KCNJ11 –
84000bp).
• Multiple mutations in these genes are implicated in Neonatal
Diabetes (NDM); for some mutations, normal activity can be
restored with Sulfonylurea drugs
K+ K+ K+
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Kir6.2 (KCNJ11)
Membrane
 Glucose   ATP
SIR1 (ABCC8)
Sulfonylurea
Normal Insulin Decreased Insulin (hyperglycemia) Normalized Insulin

20. Sanger Sequencing of KCNJ11 & ABCC8
Loss of
function/inactivatin
g G111R
causing recessively
inherited congenital
hyperinsulinism
(inherited from
asymptomatic
father)
Gain of function
R826W causing
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transient
neonatal
diabetes
(inherited from
asymptomatic
mother)
• One month old baby with NDM
(very high sugar)
• Initially managed with 2 insulin
injections per day
• Sanger Sequencing
• Detected two missense mutations
in ABCC8 gene G111R and
R826W
• Switched to oral sulfonylurea
(glibenclamide). Responded well

21. NGS Panel for Neonatal Diabetes
• Sanger sequencing of ABCC8 and KCNJ11 is laborious,
expensive (~ $2000) and requires long turn-around times
• Cases of suspected NDM with no detected mutations in
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ABCC8 and KCNJ11
• Development of NGS panel with several additional genes
involved in neonatal diabetes/hyperinsulinism
ABCC8 KCNJ11 GLUD1 GCK HADH INS HNF1A HNF4A HNF1B INS PDX1
PTF1A NEUROD1 NEUROG3 RFX6 EIF2AK3 FOXP3 GLIS3 SLC19A2 SLC2A2
IER3IP1 ZFP57 WFS1 GATA6 GATA4 CEL PAX4 BLK KLF11 LMNA PPARG
• Panel has allowed for relatively rapid and cheaper testing for
NDM and detection of new mutations in ABCC8, EIF2AK3,
GATA6, GCK, PDX1, SLC19A2

22. EGFR mutations in Lung Cancer
• Epidermal growth factor receptor (EGFR) can be mutated in
certain Non Small Cell Lung Cancers (NSCLC) and other
cancers
• Tyrosine kinase inhibitors (TKIs) (Gefitinib and Erlotinib) can
act on some mutated forms of EGFR
• Mutations usually detected from formalin fixed paraffin
embedded (FFPE) blocks by Sanger Sequencing or PCR
based methods
• Sanger sequencing has limited (~ 20%) sensitivity
• PCR methods need you to know the mutations a priori
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23. EGFR mutations by NGS
• Ultra-deep sequencing of EGFR from FFPE samples can
detect mutations at sensitivities up to 0.1%
• Somatic mutation NGS panels can be used to detect
mutations not just in the hotspots of certain genes, but over
entire sequences of multiple genes (e.g. KRAS, NRAS,
BRAF)
• Research projects underway to determine the clinical
relevance of EGFR mutations detected from circulating tumor
DNA in plasma
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24. BCR-ABL1 Fusions in CML
• Chronic Myelogenous Leukemia characterized by reciprocal
translocation of chromosomes 9 & 22 resulting in the
Philadelphia chromosome
• Fusion of Abelson (ABL1) proto-oncogene on chr 9 with the
breakpoint cluster region gene (BCR) on chr 22 , generating
the constitutively active BCR-ABL1 fusion oncogene
• Responds to tyrosine kinase inhibitor (TKI) – Imatinib
• Need to monitor minimal residual disease (MRD)
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25. BCR-ABL1 Testing in CML by RQ-PCR
Reverse transcribe RNA to DNA using Random Hexamers + TaqMan DNA quantitation
14
12
10
8
6
4
2
BCR-ABL ABL
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Tube Ct* Calculated Conc
(copies/uL)
Tube Ct* Calculated Conc
(copies/uL)
Standard 101 copies 32.97 9.8 Standard 101 copies 34.60 11
Standard 102 copies 29.44 104.8 Standard 102 copies 31.29 97
Standard 103 copies 26.12 977 Standard 103 copies 27.84 1017
Patient Major BCR-ABL 33.13 6.7 Patient ABL 26.45 5240
Major BCR-ABL/ABL Ratio is 0.13%
0
0 10 20 30 40
Log of target concentration

26. Digital PCR for BCR-ABL1 in CML
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dA dT dG dC
Polymerase + Nucleotides +
Primers + Probes
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• Droplet digital RT-PCR
• Two color droplet detection of
BCR-ABL1 fusion gene and
ABL1 as reference gene
• Absolute count as against
relative quantitation
• No need for a standard curve
• Lower limit of detection (LOD)
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27. Challenges and Questions
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28. Accessibility and Cost (India vs. US)
• US Population – 316 Mn,
17% rural
• Per capita income (PPP) - $
44800
• NGS machines: ~1000
• Physicians/ 1000 people:
2.45
• Public health expenditure (%
of GDP) – 8.3%
• Total health expenditure (per
capita) - $8233
• Established insurance system
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• India Population – 1.25 Bn,
68% rural
• Per capita income (PPP) –
$ 5350
• NGS machines: ~100 (80% in
research)
• Physicians/ 1000 people: 0.7
• Public health expenditure (%
of GDP) – 4.0%
• Total health expenditure (per
capita) - $126
• Out of pocket payment
Sources: WHO (2010), World Bank (2013)

29. Awareness and Education
• Limited exposure to genetics and molecular biology
during medical training
• Mandatory CMEs only recently introduced
• Limited ability to select appropriate tests and interpret
test results for optimal patient management
• Usage of molecular diagnostics guided decision making
is limited to very few conditions (e.g. Imatinib in CML,
Traustuzumab in Breast Cancer, Sulphonylurea in NDM)
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30. This presentation is the property of
Other Challenges
• Complex data interpretation & data storage
requirements
• Variants of unknown significance (VUS)
• Limited databases for ethnicities
• Incidental findings
• Ethical considerations

31. Questions that need to be answered
• Is the test relevant? Could it’s results alter the course of
treatment?
• What are the criteria for testing?
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• Are the results actionable?
• Will the results be reliable?
• Will the results be available in time?
• Will the results be interpretable?
• Is the test affordable? Is the follow-up treatment affordable? Is
the test more expensive that the treatment?

32. This presentation is the property of
Some solutions
• Increased availability of economical tests
• Availability and selection of relevant and actionable tests
• Multiplexing of tests and samples to reduce costs
• Reporting of only relevant and actionable data from panels
• Quick turn around time
• Clear communication with the clinicians

33. Thank You
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