Precision Medicine and Next Generation Sequencing: Practical Applications John Pfeifer, MD, PhD Washington University School of Medicine Department of Pathology

Preliminary ADASP Program (Oct. 2007)

Preliminary ADASP Program (Jan. 2008)

Many of the perceived challenges mix together diagnostic, prognostic, and therapeutic implications, which fosters uncertainty and anxiety “We have recently entered a transition period in which specific genetic knowledge is becoming critical to the delivery of effective health care for everyone.” “We have recently entered a transition period in which specific genetic knowledge is becoming critical to the delivery of effective health care for everyone.” Guttmacher AE, et al. N Engl J Med 2002;347: Guttmacher AE, et al. N Engl J Med 2002;347: “… the impending revolution in molecular and computational biology is comparable with previous periods of sweeping change in diagnostic pathology.” “… the impending revolution in molecular and computational biology is comparable with previous periods of sweeping change in diagnostic pathology.” Finn WG. J Mol Diagn 2007;9:

Patient Care Growing complexity of recognized genetic aberrations characteristic of specific diseases offers opportunities to develop new clinical molecular testing paradigms Growing complexity of recognized genetic aberrations characteristic of specific diseases offers opportunities to develop new clinical molecular testing paradigms Growing complexity of molecular testing paradigms offers opportunities for defining indications for testing and test interpretation Growing complexity of molecular testing paradigms offers opportunities for defining indications for testing and test interpretation Growing demand creates opportunities for possible new revenue streams Growing demand creates opportunities for possible new revenue streams

Research opportunities “Considering the fact that cell function is controlled by a complex network of functionally active signaling pathways, it is unlikely that expression analysis of a single or small number of proteins will precisely predict the clinical outcome of an individual tumor.” “Considering the fact that cell function is controlled by a complex network of functionally active signaling pathways, it is unlikely that expression analysis of a single or small number of proteins will precisely predict the clinical outcome of an individual tumor.” Dietel M, et al. Virchows Arch 2006;448:

Opportunities for Education: Residents and Fellows Community hospital pathologists were recently asked to identify the skills and knowledge they expect of a newly minted pathologist: Since… Since… many groups are doing at least PCR, FISH, ISH, and cytogenetics many groups are doing at least PCR, FISH, ISH, and cytogenetics clinicians are demanding molecular testing clinicians are demanding molecular testing Groups depend on young pathologists to… Groups depend on young pathologists to… bring these techniques with them from the university bring these techniques with them from the university know the indications for testing know the indications for testing interpret the results interpret the results Horowitz RE. Hum Pathol 2006;37:

Pathology Microarrays Cytogenetics Next Gen Sequencing Sanger Sequencing Sanger Sequencing Biomedical Informatics Biomedical Informatics

Comprehensive Cancer Set Have an established role in patient care for diagnosis, prognosis, or therapy Comparable performance with FFPE and frozen samples Analysis of small biopsy & cytology specimens Detect variants with as little as 20% tumor cellularity 1000x fold sequence read depth fold sample multiplexing to reduce cost Comprehensive informatics and interpretive reporting Currently reimbursed by Sanger methodology Requirements

Practical Applications: The first thing practicing pathologists need to know is that there is no need for anxiety Only about 6% of cases need molecular testing Only about 6% of cases need molecular testing In most cases tested, morphology is required to establish the need for molecular analysis In most cases tested, morphology is required to establish the need for molecular analysis In most cases tested, morphology is required to identify tissue to be evaluated In most cases tested, morphology is required to identify tissue to be evaluated Even in cases tested, molecular analysis can not currently be employed to establish stage (depth of invasion) or is untenable as a routine method for establishing stage (status of all lymph nodes) Even in cases tested, molecular analysis can not currently be employed to establish stage (depth of invasion) or is untenable as a routine method for establishing stage (status of all lymph nodes) Even in cases tested, molecular analysis can not be used to evaluate the margins of excision Even in cases tested, molecular analysis can not be used to evaluate the margins of excision Need more Pathologists than are available Need more Pathologists than are available

CAP, AMP, ACMG, and so on, have committees working in the personalized medicine landscape CAP, AMP, ACMG, and so on, have committees working in the personalized medicine landscape TRIG and other non-institutional working groups are developing teaching materials TRIG and other non-institutional working groups are developing teaching materials The CAP checklist for NGS was released summer 2012; the ACMG guidelines for NGS were just released for comment The CAP checklist for NGS was released summer 2012; the ACMG guidelines for NGS were just released for comment Published guidelines are being developed that address the required fund of knowledge

The American Board of Pathology has yet to provide guidance Section III Cognitive Expertise of the ABP’s “Maintenance of Certification Booklet of Information” states that: E. The ABP recognizes the breadth of pathology practice. 1. The primary examinations (AP, CP, AP/CP) will be modular and the diplomats will be able to select modules that are as relevant as possible to individual practice settings. 2. The subspecialty MOC examinations in Hematology, Molecular Genetic Pathology, Neuropathology, and Pediatric Pathology will be modular. The remainder of the subspecialty examination will consist of 150 questions covering the general practice of the subspecialty. 3. For both primary and subspecialty exams, all modules will be graded together as one examination. 4. See “MOC Presentation” on the MOC section of the ABP Web site for a proposed list of modules. These are subject to change prior to administration of the first examination in F. The exact nature of the modules to be provided is under development…

Pathology Assessment Accessioning DNA Isolation Pathology Assessment Accessioning DNA Isolation Tissue Sample Submission DNA Sample Submission Library Submission Data Submission Library Preparation Library Preparation Sequencing Bioinformatic Analysis Tier 1 Base Calling Alignment Variant Calling Tier 2 Annotation Knowledgebase Tier 3 Clinical Report Bioinformatic Analysis Tier 1 Base Calling Alignment Variant Calling Tier 2 Annotation Knowledgebase Tier 3 Clinical Report Clinical Samples

Practicing pathologists who do genomic pathology need some level of technical and genetic expertise Familiarity with different platforms (specifically those marketed by Illumina and Life Technologies) Familiarity with different platforms (specifically those marketed by Illumina and Life Technologies) Differences in turn around time Differences in turn around time Differences in error rate Differences in error rate Differences in capacity Differences in capacity Understanding of the differences in hybridization-based approaches versus amplification-based approaches Understanding of the differences in hybridization-based approaches versus amplification-based approaches Use of panels, versus exomes, versus genomes Use of panels, versus exomes, versus genomes Subspecialty training in molecular pathology Subspecialty training in molecular pathology

Practicing pathologists who do genomic pathology also need some level of bioinformatic expertise Implications of depth of coverage for detecting germline mutations versus acquired mutations Implications of depth of coverage for detecting germline mutations versus acquired mutations Optimization and clinical validation of bioinformatic pipelines Optimization and clinical validation of bioinformatic pipelines Differences in bioinformatic approaches for SNVs, indels, CNVs, and translocations Differences in bioinformatic approaches for SNVs, indels, CNVs, and translocations Impact of nucleic acid quantity and quality on Bayesian metrics Impact of nucleic acid quantity and quality on Bayesian metrics Different -omic techniques Different -omic techniques Wood LD, et al. Science 2007;318: International HapMap Consortium. Nature 2007;449: Korbel JO, et al. Science 2007;318: West RB, et al. Lab Invest 2007;87:

Detection of indels varies by software tool: FLT3 ITD detection is an example Tested a set of 24 cases with known FLT3 ITDs by WUCaMP28 Pindel correctly identified ITDs in 23/24 cases Identified all ITDs (24/24) by de-novo assembly methods (detected allele frequencies of less than 4%) No false positive results Reference: Spencer DH et al. J Mol Diagn 2013;15:81-93 red= not detected green=detected

Pathologists will need to be familiar with a range of -omics techniques Genomics (DNA) Genomics (DNA) Transcripomics (mRNA) Transcripomics (mRNA) Interferomics (iRNA) Interferomics (iRNA) Epigenomics Epigenomics Tumor cells (oncomics) versus stroma (stromics) Tumor cells (oncomics) versus stroma (stromics) Proteins versus nucleic acids (eg, proteomics and metabolomics) Proteins versus nucleic acids (eg, proteomics and metabolomics)

Practicing pathologists who do genomic pathology will need to understand their central educational role to their clinical colleagues In a model system evaluating the use of APC testing, In a model system evaluating the use of APC testing, Only 83% of patients had valid reasons for testing (clinical features of familial adenomatous polyposis or were at risk for the disease) Only 83% of patients had valid reasons for testing (clinical features of familial adenomatous polyposis or were at risk for the disease) The appropriate strategy for pre symptomatic testing was used in 79%, only 19% received genetic counseling before the test, and only 17% provided written informed consent The appropriate strategy for pre symptomatic testing was used in 79%, only 19% received genetic counseling before the test, and only 17% provided written informed consent In 32% of the cases the physicians misinterpreted the test results In 32% of the cases the physicians misinterpreted the test results Giardiello FM, et al. N Engl J Med 1997;336:823-7

Practicing pathologists who do genomic pathology will need to understand their central educational role extends beyond their clinical colleagues to include As a source of professional information regarding so called “recreational genomics” which may cause Needless worry Needless worry Poor medical decisions Poor medical decisions Discrimination Discrimination Potential for litigation (eg, right to know, relatedness) Potential for litigation (eg, right to know, relatedness) Kaiser J. Science 2007;318:1843

A concluding note of caution… “… the four letters of the genetic code are H, Y, P, and E, and medical providers must realize that the molecular biology business is as adept at promoting its wares as is any other.” Jones S.