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Use and Value of Genetic Tests for Patient Care Review of Technologies Real life examples Sherri J Bale, PhD, FACMG Definition of Genetic Testing: The analysis of human DNA in any of its forms or related products (chromosomes, RNA, proteins) Uses of Genetic Testing: To detect disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes Genetic Tests for Constitutional Mutations Cytogenetic Tests Molecular Tests Cytogenetic Test Standard karyotype, used to look for gross chromosomal anomalies in children with development delays, congenital anomalies, mental retardation FISH, used to look at 1 or 2 specific chromosomal regions suspected by the physician BAC arrays, used to look at many (100s) chromosomal regions at once, using FISH technology CGH array, used to look at MANY (50K-200K) regions at once, and identify specifically which genes are involved in the chromosomal anomaly. Child with Multiple Congenital Anomalies and/or Autism Standard karyotype CGH array - “molecular Karyotype” Patient with Tetralogy of Fallot, suspected 22q11 deletion FISH test, 2 probes, used in baby, found deletion, and confirmed dx. Provided prognostic info to family. Parents tested by FISH, found to be negative. Provided information re: risk in future children Child with multiple anomalies and autism; no specific syndrome suspected Karyotype normal CGH array followed Karyotype. Identified deletion involving end of one arm of chromosome 3 Parents tested by FISH and dad found to be balanced carrier of the deletion Prenatal diagnosis by quantitative PCR is now possible for the family. Molecular Test – PCR for wellcharacterized mutations KRAS gene test on tumor tissue from patients with colorectal cancer Obtain tumor from patient Extract DNA; PCR, then treat with enzyme that allows visualization of the mutation Molecular Test – PCR, followed by sequencing, for identification of mutation Used to identify mutation in a patient with inherited disease Number of times PCR is done and how much sequencing is required depends on SIZE of gene, MANY UNITS. Once mutation is identified, testing of parents, sibs, other relatives for ONLY that mutation, is needed. ONE or ONLY a FEW UNITS. Molecular diagnosis of Gorlin Syndrome 13 yo child presented to dentist with a jaw cyst – surgery performed but tooth was lost. Referred to geneticist. Geneticist suspected Gorlin Syndrome Molecular diagnosis involved PCR and sequencing, 26 “units” (large gene). Mutation identified. Prognosis now known: This individual would develop many skin cancers, more jaw cysts. Regular surveillance by dermatologist and dentist allowed early identification, less expensive treatment, and good clinical outcome: Teeth were saved; Minimal damage to nose, ears, eyes Use and Value of Genetic Tests Diagnosis Prognosis Appropriate surveillance leading to early care and intervention Risk Information Enables physicians to properly care for patient Is it inherited? What is the recurrence risk in future pregnancies? Prenatal/Pre-symptomatic diagnosis Allows informed decision making, preventive care Human Genetics Trends in Testing V.M. Pratt, Ph.D., FACMG Diagnostic Laboratory Testing Driver of Healthcare Decisions Diagnostic testing is foundation of healthcare Diagnostic data yields information serving public and individual Information can help identify trends for public health Data enables physicians to care for individual patients Data facilitates new test development Continuum of diagnostic lab testing 70% of healthcare decisions based on diagnostic data From diagnosis to predictive and personalized medicine Diagnostic data increasingly is providing actionable insights physicians can use to improve patients’ healthcare outcomes Genes in the News The Basics of Genetic Testing DNA->RNA->Protein DNA Cell membrane Nucleus DNA bases mRNA Chain of amino acids Gene Protein Ribosome Gene Tests – Three Common Methods Mutation Absent Chromosome Cytogenetics DNA Molecular Genetics Protein Biochemical Genetics Mutation Present Types of Mutations Single nucleotide - traditional Deletions/Insertions – copy number variants (CNVs) Missense Nonsense (creation of stop codon) Splicing Regulatory sequences (promoter, 3’ end) In frame frameshift Expansions (triplet repeat disorders) Epigenetic (methylation) Translocations and inversions Alleles Quiet! I’ll speak for both of us! Dominant Allele I’ll have to be in charge now! Normal Allele Recessive Allele Damaged Allele Human Chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Gene Mutations Mismatch Insertion Deletion Deletion General Principles Hereditary disorders can affect multiple organs Penetrance can be influenced by modifiers: genes + environment Complexity of mutational spectrum varies Different Genes – Different Functions Bone Cell Pancreas Cell Brain Cell Disease Inheritance Is Complex Gene Changes in Cystic Fibrosis Mucus Production Gene Normal Mutation 1 Mutation 2 Mutation 3 No Symptoms Severe Symptoms Mild Symptoms No Symptoms Why is Genetic Testing ordered? For couples who are having difficulty conceiving For couples who have experienced two or more miscarriages To make a diagnosis in an affected individual To see if pregnancy is at an increased risk for a genetic disorder To test people with a family history of a specific inherited disease; patients may want to know if they might develop the disease or pass it on to their children Continuum of Diagnostic Lab Testing Diagnostic Predictive Personalized ► Confirm diagnosis ► Tailor drug treatment to genotype -Cystic Fibrosis ► Determine higher chance for disease before symptoms appear -HIV Therapy -Huntington Disease Physician Patient Pharma Common genetic disorders Inherited (predictive or diagnostic) Acquired (predictive or diagnostic) Cystic fibrosis Thrombophilia Hereditary hemochromatosis Fragile X syndrome Chronic myelogenous leukemia (CML) Pharmacogenetics (personalized) Cytochrome P450s HLA New Assay/Biomarker Progression Evidence-based medicine Retrospective clinical trials Clinical Research Biomarker associated with disease Prospective clinical trials Test Translation Clinical Validity Clinical Utility Lab test developed Analytical validation Test can predict clinical outcomes Benefits patients Emerging Technologies and Testing The genome is complex High throughput DNA sequencing microRNAs Copy Number Variants (CNVs) Epigenetics methylation Proteomics Up and down regulation Disease-specific patterns New high throughput DNA sequencing methods 454 (Roche) 20 megabases per 4.5-hour run capable of detecting mutations in an amplicon pool at low sensitivity Reads: ~100 base pairs Can’t read highly repetitive or long polymers Solexa (Illumina) Can sequence through homopolymers and repetitive sequences 10's of millions of short (24-36 bp) reads Single-end vs. paired end reads MicroRNAs (miRNA) Single-stranded RNA molecules 21-23 nt Transcribed from non-coding DNA Regulate gene expression Cancer May enable classification of cancers (CUP) Determine therapy Copy Number Variants (CNVs) (Variome) Large deletions or duplications of DNA Usually cannot be detected by DNA sequencing Newer technologies aCGH Impacts Autism Alzheimer disease Parkinson disease susceptibility to HIV-1 some forms of color blindness Array CGH Cross-platform identification and validation of CNVs Jennifer L. Freeman et al. Genome Res. 2006; 16: 949-961 Epigenetics Changes in chromatin structure (how DNA is packaged) or alters gene activity without changing the DNA DNA methylation Modification of histones Position effects Cancer and imprinting disorders Genetic Tests Find Mutations, NOT Disease Chances of Developing Breast Cancer by Age 65 100 10 90 9 80 8 70 7 60 6 50 5 40 4 30 3 20 2 10 1 0 Altered BRCA1 0 Normal BRCA1 Benefits of Gene Testing • • • • Relief Fewer Checkups Informed Decisions Intervention Pharmacogenetics V.M. Pratt, Ph.D., FACMG Personalized Medicine/Pharmacogenetics Getting the right dose to the right patient at the right time Chronic Myelogenous Leukemia (CML) example Philadelphia chromosome t(9;22) Detectable by standard cytogenetics (karyotype analysis) FISH Fusion of BCR/ABL genes FISH Quantitative PCR Treatable with Gleevac Some people become resistant to Gleevac Mutations in tyrosine kinase domains (DNA sequencing) Example: Tamoxifen Antiestrogen Used to treat estrogen receptor (ER)+ breast cancer Metabolized by cytochrome P450 2D6 blocks the activity of estrogen which can stop the growth of some breast tumors. Endoxifen (active form of Tamoxifen) MEDCAC reviewing if should pay for Medicare population FDA reviewing whether to revise the drug label to recommend CYP450 2D6 (CYP2D6) testing Tamoxifen use treat breast cancer that has metastisized. treat early breast cancer in women who have already been treated with surgery, radiation, and/or chemotherapy. reduce the risk of developing a more serious type of breast cancer in women who have had ductal carcinoma in situ (DCIS) and who have been treated with surgery and radiation. reduce the risk of breast cancer in women who are at high risk for the disease due to their age, personal medical history, and family medical history. Tamoxifen Metabolism Various Assays/Platforms for CYP2D6 Platform Legal Status Autogenomics INFINITI Luminex xTag RUO ParagonDx IUO RUO Roche Amplichip FDA-cleared ABI SNaPshot LDT Example: Clopidogrel (Plavix) inhibit blood clots in coronary artery disease, peripheral artery disease, and cerebrovascular disease. Metabolized by cytochrome P450 2C19 to active form 2-{1-[1-(2-chlorophenyl)-2-methoxy-2oxoethyl]-4-sulfanyl-3-piperidinylidene}acetic acid FDA-recently announced that clopidogrel cannot be taken with Prilosec (omeprazole) and Nexium (esomeprazole) Inhibitors of 2C19 Clopidogrel use Prevention of vascular ischemic events in patients with symptomatic artherosclerosis Acute coronary syndrome without STsegment elevation (NSTEMI) ST elevation MI (STEMI) It is also used, along with aspirin, for the prevention of thrombosis after placement of intracoronary stent Various Assays/Platforms for CYP2C19 Platform Legal Status Autogenomics INFINITI Luminex xTag RUO Roche Amplichip FDA-cleared ABI SNaPshot LDT IUO Laboratory Procedures and Coding Presented by Kaye Jones, MLS(ASCP), CPC Jan. 13, 2010 Objective To improve your understanding of molecular diagnostic CPT coding Molecular Codes CPT codes 83890-83914 represent steps performed during molecular diagnostic procedures. CPT codes are assigned based on the different steps and the number of times each type of step is performed. Example: 83898 Amplification x3 83896 Nucleic acid probe, each x25 Molecular Codes Different molecular methods may be used for the same analyte. Those methods may vary among different laboratories. Therefore, all labs will not necessarily suggest the same CPT codes for the same test. Molecular Codes Another concern is that there are no clear written guidelines for how to assign units of service. For example: When a Cystic Fibrosis procedure for 23 mutations is performed by two different labs One lab bills for 23 probes (1 per mutation). Whereas, the other lab bills for 46 probes (2 per mutation because you need a normal and a mutant probe for each in order to interpret the assay). Molecular Codes All stakeholders within the industry recognize the issues surrounding the assignment and billing of molecular diagnostic procedures. These concerns have resulted in a number of organizations and entities working together to review and address the problems. Cytogenetics This is another area where CPT codes are assigned based on the procedural steps performed. Routine chromosome analysis typically requires three steps to complete the procedure. 88230 88262 88291 picture Tissue culture Karyotyping Interpretation and report with a of the actual chromosomes Cytogenetics Cytogenetic FISH is often necessary to diagnose constitutional defects. Cytogenetic FISH may also be a stand alone order necessary to evaluate leukemias such as CML and MM. Units of service are determined by the number of FISH probes and specific procedures needed to evaluate the cells. Molecular Diagnostics and Cytogenetics The newer CGH procedure can be thought of as a combination of molecular diagnostic procedural steps needed to prepare the patient’s sample and the cytogenetic CGH chip analysis. There is much confusion within the industry regarding how to assign CPT codes to CGH procedures. Molecular Diagnostics and Cytogenetics Array CPTs 88384-88386 are in the Surgical Pathology section of CPT. These codes are global codes with TC and Professional components, only represent the work needed for the array chip and I&R, sample preparation is coded with molecular diagnostic codes, and only appropriate for when a physician/pathologist performs the I&R. Molecular and Cytogenetic Codes In summary: Molecular procedures are coded based on the procedural steps. Units of service are determined by the number of times each step is performed. Different procedures may exist for the same analyte, which makes the CPT coding different. Lack of standardized coding guidelines add to the complexities of how to assign CPTs. Genetics Questions?