Clinical molecular diagnostics for drug guidance
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The presentation discusses the role of molecular diagnostics in clinical decision making, highlighting next-generation molecular diagnostic techniques and their utility, particularly in resource-limited settings. It covers various applications, such as pathogen identification, predictive testing, and oncology, while addressing challenges like accessibility, cost, and interpretation of genetic data. Finally, it emphasizes the need for economical tests, actionable data, and improved clinician communication to enhance the use of molecular diagnostics in patient care.
Clinical molecular diagnostics for drug guidance
- 1. Utility of Molecular Diagnostics in clinical decision making Nikhil Phadke, PhD GenePath Dx, Pune, India This presentation is the property of
- 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? This presentation is the property of 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 This presentation is the property of
- 8. Traditional Capillary/Sanger Sequencing Chromatogram + - Shorter Longer fragments fragments This presentation is the property of 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 This presentation is the property of 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) This presentation is the property of 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 This presentation is the property of 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) This presentation is the property of
- 13. Digital PCR – An evolution of PCR Conventional PCR Real-time PCR Digital PCR Real time detection in closed system High Low This presentation is the property of 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 This presentation is the property of
- 15. Molecular diagnostics across medical disciplines This presentation is the property of 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) This presentation is the property of
- 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 This presentation is the property of 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 This presentation is the property of 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+ This presentation is the property of 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 This presentation is the property of 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 This presentation is the property of 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 This presentation is the property of
- 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 This presentation is the property of
- 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) This presentation is the property of
- 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 This presentation is the property of 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 This presentation is the property of dA dT dG dC Polymerase + Nucleotides + Primers + Probes + - + - + + + + + + - + + - + - + + + + + + - + • 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) + + + + + + + + + + + + + + + + + +
- 27. Challenges and Questions This presentation is the property of
- 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 This presentation is the property of • 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) This presentation is the property of
- 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? This presentation is the property of • 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 This presentation is the property of
Editor's Notes
- #3: The basics Examples to illustrate Challenges and questions raised Some take home messages
- #11: Fragmentation / targeted amplification Addition of Priming and Multiplexing Adapters / Barcodes (ligation or PCR) Clonal amplification of each fragment (Emulsion PCR or Bridge PCR) Physical separation of each clonally amplified fragment through droplets captured in picotiter plates or on the surface of a flow cell. Real-time sequencing by sequential addition of one nucleotide at a time, or through the use of flurescently labeled reversibly terminated nucleotides Data acquisition through a cascade reaction that either produces an H+ ion (nanofabricated pH meter) or a bioluminescence event (luciferase) or through fluorescence monitoring of the flow cell Conversion into nucleotide base calls. Assembly of fragment data to final form.
- #12: Polymerase anchored at base of Zero-Mode Waveguide. All nucleotides fluorescently labeled Detection of single nucleotide being incorporated by polymerase in real-time Large arrays of ZMWs 5) DNA unwound and single stranded DNA ratcheted through protein nanopores embedded in artificial substrate 6) DNA bases detected as strand is stalled based on conductivity across the nanopore 7) Large arrays of nanopores
- #14: Microreactors in digital PCR. Much like the emulsion PCR sample prep for NGS. From Qualitative results to relative quantification to absolute quantification (Poisson statistics)
- #17: 1) Frequency of screening. Type of screening (e.g. alternating mammogram & MRI), Age of initial screening note mammo slightly less sensitive for BRCA1 due to higher breast density 2) e.g. Salpingo-oophorectomy after childbearing, bilateral mastectomy, chemoprevention like anti-estrogen therapy for BRCA2 mutation carriers 3) e.g. avoidance of radiation for TP53 mutation carriers, gene-specific treatment options like PARP inhibitors and platinum salts for BRCA1 carriers. 4) Evaluation of risk and testing of other family members 5) Evaluate the option on pre-implanatation genetic testing
- #18: Tumor Supressor genes. Large genes. BRCA1 (81kb/24 exons/1863amino acids), BRCA2 (84kb/26 exons/3418 amino acids). Nearly 800 clinically relevant mutations reported. Laborious and expensive to sequence. ~ $3500/sample. Patent related complications.
- #19: Cheaper and quicker way to sequence BRCA1 & 2 Several additional genes included in the panel (the one’s in bold – PALB2 and MEN1 were recently reported as important in NEJM articles)
- #20: Sulphonylurea Receptor (SUR1) and Inwardly Rectifying potassium channel (Kir6.2) - 4 subunits of each form the Katp channels. In pancreatic beta cells high glucose levels lead to increased ATP production, Channels sense the intracellular ATP levels and close. This causes increased membrane potential which opens voltage dependent calcium channels which trigger insulin exocytosis, Activating mutations can lead to neonatal diabetes
- #23: FFPE DNA extraction is hard, and sometimes poor quality DNA Tumor tissue is heterogenous mass along with normal tissue. Low level mutations can be easily missed 3) Sanger has low sensitivity, and PCR based methods need prior knowledge. Looking for all mutations by PCR can be laborious and expensive.
- #29: Often get patients who simply cannot afford the tests. In many of those cases, even if the cost of the test were removed from the equation, the patient would not be able to afford the cost and burden of treatment In some cases (especially infectious diseases) the cost of the test overwhelms the cost of the treatment, and the accessibility to the treatment, so it becomes easier to try a variety of treatments rather than test guided therapy
- #31: Large storage requirements. Need computaional horsepower, and fast internet connectivity. Also need data management and interpretation expertise How do you handle large numbers of variants of unknown significance. Up to 1200 VUS in a single exome sequencing Most data has been generated on Caucasian populations. Limited data available for Indians and other ethnic groups (further complicated by heterogenous populations). This exacerbates the VUS problem further. How do you report incidental finding i.e. something that you were not looking for in the first case? How do you manage the data, it’s privacy. How do you deal with findings that might impact other family members?
- #32: From the perspective of the clinician Is the test affordable? Is it more expensive than the treatment? Is the treatment affordable? Is the test relevant? Are the results actionable? Will the results be reliable? Will the results be available in time? From the perspective of the diagnostician Is there sufficient awareness about the need for the test? Do the clinicians have the knowledge and ability to interpret the results of the tests?
- #33: Availability of relevant tests include the ability of data for the ethnicities that are being tested