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The Journal of Molecular Diagnostics, Vol. 17, No. 2, March 2015 jmd.amjpathol.org SPECIAL ARTICLE Reporting Incidental Findings in Genomic Scale Clinical SequencingdA Clinical Laboratory Perspective A Report of the Association for Molecular Pathology Madhuri Hegde,*yz Sherri Bale,*x Pinar Bayrak-Toydemir,*{k Jane Gibson,*,** Linda Jo Bone Jeng,*yy Loren Joseph,*zz Jordan Laser,*xx Ira M. Lubin,*{{ Christine E. Miller,*k Lainie F. Ross,*kk*** Paul G. Rothberg,*yyy Alice K. Tanner,*yz Patrik Vitazka,*x and Rong Mao*{k From the Incidental Findings Working Group of the Association for Molecular Pathology (AMP) Clinical Practice Committee and the Whole Genome Analysis Working Group,* Bethesda, Maryland; the Department of Human Genetics,y Emory University School of Medicine, Atlanta, Georgia; the Emory Genetics Laboratory,z Emory University, Decatur, Georgia; GeneDx,x Gaithersburg, Maryland; the Department of Pathology,{ University of Utah School of Medicine, Salt Lake City, Utah; the Department of Molecular Genetics,k ARUP Laboratories, Salt Lake City, Utah; the Department of Clinical Sciences,** University of Central Florida College of Medicine, Orlando, Florida; the Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, the Department of Pathology, and the Division of Human Genetics, Department of Pediatrics,yy University of Maryland School of Medicine, Baltimore, Maryland; the Departments of Pathologyzz and Pediatrics,kk University of Chicago, Chicago, Illinois; the Division of Cytogenetics and Molecular Pathology,xx North Shore Long Island Jewish Health System, New Hyde Park, New York; the Division of Laboratory Programs, Standards, and Services,{{ Centers for Disease Control and Prevention, Atlanta, Georgia; the MacLean Center for Clinical Medical Ethics,*** University of Chicago, Chicago, Illinois; and the Department of Pathology and Laboratory Medicine,yyy University of Rochester School of Medicine and Dentistry, Rochester, New York Accepted for publication October 21, 2014. Address correspondence to Madhuri Hegde, Ph.D., Department of Human Genetics, 615 Michael St., Whitehead Biomedical Research Bldg, Emory University School of Medicine, Atlanta, GA 30022. E-mail: [email protected]. Advances in sequencing technologies have facilitated concurrent testing for many disorders, and the results generated may provide information about a patient’s health that is unrelated to the clinical indication, commonly referred to as incidental findings. This is a paradigm shift from traditional genetic testing in which testing and reporting are tailored to a patient’s specific clinical condition. Clinical laboratories and physicians are wrestling with this increased complexity in genomic testing and reporting of the incidental findings to patients. An enormous amount of discussion has taken place since the release of a set of recommendations from the American College of Medical Genetics and Genomics. This discussion has largely focused on the content of the incidental findings, but the laboratory perspective and patient autonomy have been overlooked. This report by the Association of Molecular Pathology workgroup discusses the pros and cons of nextgeneration sequencing technology, potential benefits, and harms for reporting of incidental findings, including the effect on both the laboratory and the patient, and compares those with other areas of medicine. The importance of genetic counseling to preserve patient autonomy is also reviewed. The discussion and recommendations presented by the workgroup underline the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and laboratories. (J Mol Diagn 2015, 17: 107e117; http://dx.doi.org/10.1016/j.jmoldx.2014.10.004) The Incidental Findings Working Group is a joint working group of the AMP Clinical Practice Committee and the Whole Genome Analysis Working Group. The 2013 Clinical Practice Committee consisted of: Matthew Bankowski, Milena Cankovic, Jennifer Dunlap, Larissa Furtado, Jane Gibson, Jerald Gong, Thomas Huard, Linda Jeng, Loren Joseph (Chair 2013 to 2014), Marilyn Li, Paul Rothberg, M. Fernanda Sabato, and Patrik Vitazka. The 2013 Whole Genome Analysis Working Group consisted of: Nazneen Aziz, Pinar Bayrak-Toydemir, Daniel Farkas, Jane Gibson (Chair), Bert Gold, Andrea Ferreira-Gonzalez, Timothy Greiner, Loren Joseph, Roger Klein, Debra Leonard, Marilyn Li, Ira Lubin, Elaine Lyon, Copyright ª 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.10.004 Karen Mann, Rong Mao, Victoria Pratt, Iris Schrijver, and Patrik Vitazka. Disclosures: M.H. is on the Scientific Advisory Board for Ingenuity/ Qiagen and Oxford Gene Technologies; S.B. is on the Scientific Advisory Board for Ingenuity/Qiagen and Raindance Technologies; Standard of practice is not defined by this article, and there may be alternatives. See Disclaimer for further details. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Hegde et al The introduction of new genomic technologies in clinical medicine,1 particularly next-generation sequencing (NGS) of entire exomes and genomes, generates an abundance of data only some of which is relevant to the clinical question that prompted the investigation.2 Analysis of evidence that is based on targeted gene panels can also result in the generation of data not relevant to the clinical question posed because some genes are associated with multiple medical conditions.3e5 These additional findings present difficulties to the laboratory, most importantly deciding which information should be reported. A number of factors go into this decision, including patient preferences usually communicated through the consent process and documented on the consent form, the clinical relevance of findings, the potential for false-positive or -negative results, and the application of professional practice standards. In addition, the laboratory must grapple with the issues of either confirming potentially important incidental findings or reporting results with a lower quality threshold than is usually required.3 Finally, if the laboratory does have a policy of reporting potentially important incidental findings, a predictable unintended consequence could be that the ordering provider misinterprets a report with no pathogenic variants to mean meaning that there are actually no pathogenic variants in a specific gene, when, in fact, the gene sequence and its disease-causing mutation spectrum may not have been fully covered during sequencing; therefore, pathogenic variants could have been missed.6 This document was prepared by a Joint Working Group of the Association for Molecular Pathology Clinical Practice Committee and Whole Genome Analysis Working Group to discuss these issues from a laboratory perspective and to develop recommendations for laboratories considering policies for reporting incidental findings. By way of definition, the term incidental finding is used in this report to include all findings that are clearly or expected to be pathogenic but do not address the clinical question that motivated testing. This is the same intent as was used in the recently published policy statement from the American College of Medical Genetics and Genomics (ACMG).7 The ACMG Working Group on the reporting of incidental findings from clinical exome and genome sequencing recommended that incidental findings for 56 genes that are clearly pathogenic and clinically actionable be reported by the laboratory. For example, identifying patients with an inactivating mutation in the APC gene that causes familial adenomatous polyposis permits increased surveillance and early detection of cancerdwhen it can be best treated. The ACMG document urged that this policy apply regardless of the age of the patient, except for fetal samples, and also included any germline testing done as part of cancer-related genomic scale sequencing. The ACMG document is a significant milestone in the development of clinical genomic sequencing services because it clearly defines the issues and challenges and makes a major contribution to setting policies in this area. An ACMG workgroup recommendation specifically excludes giving the patient the opportunity to 108 refuse or opt-out of receiving a report that details pathogenic mutations identified in these 56 genes, catalyzing much discussion in the field.3,8e13 The goals of the ACMG report, the subsequent discussions, and this document are to inform the development of policies which maximize the benefits and minimize the harms that can result as a consequence of the reporting of incidental findings to inform clinical and personal health care decisions. Here, we discuss several issues in the reporting of incidental findings in clinical genomic sequencing from the laboratory perspective. In particular, we explore the potential benefits and harms that could result from different decisions for reporting incidental findings, including the effect on both the laboratory and the patient. We consider the relation between the reporting of incidental findings in genomics and other areas of medicine. We also address how to include patient decision making in the testing process to preserve respect for patient autonomy. We make suggestions about how to shape the pretest counseling/informed consent process and the laboratory report to accurately convey what was and was not found. Finally, this document emphasizes the need for continued research and discussion among all stakeholders to improve our understanding of the effect of different policies on patients, providers, and laboratories. From Sanger Sequencing to NGS Sanger sequencing is used routinely by clinical laboratories for diagnosis of heritable conditions and to detect somatic mutations in cancer. Although Sanger sequencing is considered the gold standard, its relatively low throughput and high cost have been major obstacles to its use in sequencing large regions of the human genome in the clinical setting. Thus, NGS, also called massively parallel sequencing, has become an attractive alternative for applications that require assessment of multiple genes, exomes, or genomes in situations in which the clinical presentation is ambiguous and/or previous diagnostic testing did not lead to identification of the underlying molecular basis for the patient’s clinical presentation.1,14e17 In general, genomic DNA used for library preparation for NGS can be either used with (panels and exomes) or without (genomes) enrichment. Each of the target enrichment approaches has advantages and drawbacks. For example, all enrichment procedures can suffer from analytical limitations, including but not limited to the sequencing of pseudogenes, variation of amplification efficiency across numerous targets, GC-rich area biases, and allelic drop out. For the sequencing of a gene panel, lowcoverage or low-quality segments can be resolved by analysis with alternative methods including Sanger sequencing, either preemptively or after such low-coverage regions are found during review of sequencing data. Resolving all lowcoverage segments for whole-exome sequencing (WES) is, however, highly impractical.18 WES is considered to be a screening tool because not all genes are covered equally well jmd.amjpathol.org - The Journal of Molecular Diagnostics Reporting Incidental Findings in Genomics and some genes or exons are not covered at all (eg, SMN1 gene and triplet repeat disorders). WES has less than optimal analytical sensitivity but offers relatively high clinical sensitivity. Although whole genome sequencing (WGS) has gained popularity in the research area, because of its high associated costs for sequencing and data storage, it is often performed at a significantly lower coverage than the WES, reducing its technical sensitivity. Another advantage of WES is the relative ease of interpretation of variants because WES targets only the exome (ie, approximately 92% of all known coding exons and exon-intron boundaries, approximately 180,000 exons of approximately 20,500 genes), which represents approximately 1% to 2% of the total human genome (or approximately 30 Mb of sequence). Therefore, WES represents a compromise between the number of genes that are assessed (all known genes), albeit at imperfect coverage, and a limited number of well-covered genes, compared with more analytically sensitive/specific panels and WGS. exceptions apply. Much more difficult to interpret are missense variants, especially those which are not located in evolutionarily well-conserved regions, not found in certain functional motifs or domains, or not known to be in residues that are subject to posttranslational modification. In addition, variants for which functional predictions with the use of programs such as Sorting Intolerant from Tolerant and Polymorphism Phenotyping, favor indeterminate or benign variants that result in a conservative change, or variants outside of the canonical splice site for which splicing predictors are mixed are also difficult to interpret. Erroneous entries in public databases that are not curated or clinically validated, insufficient evidence because of poorly designed scientific studies (insufficient functional evidence or underpowered segregation analyses), isolated reports of association of a particular gene with a phenotype or presence of a phenotype that has not been previously associated with the gene, and lack of clinical indications/notes can also hinder interpretation of identified variants.20 Interpretation of Genomic Variants Genome-wide sequencing approaches usually yield tens of thousands variants when the sequenced data are aligned to the reference genome. Not all variants are pertinent to the patient’s clinical presentation and, in fact, usually only one (for dominant disorders) or two variants (for recessive disorders) cause disease.19 It is impossible to evaluate every single variant manually; therefore, bioinformatics analysis must be used to sort (filter) variants to narrow the number of relevant candidates. In the filtering process, the variants are assessed with several considerations, including population frequency of the variant, presence of the variant in clinical or sequencing databases such as Human Gene Mutation Database, ClinVar, Online Mendelian Inheritance in Man, National Heart, Lung, and Blood Institute Grand Opportunity Exome Sequencing Project, and Locus-Specific Databases; likely mode of inheritance; phenotypic features and genes associated with these features; list of genes likely to explain the clinical presentation; predicted functional severity of the sequence change; variant type such as synonymous, missense, nonsense, frameshift, inframe; and pathogenic versus benign variants (ie, variants in internal databases that violate the Hardy Weinberg Equilibrium, variants present in unaffected homozygotes); tissue expression of a gene with a variant; relevance to the clinical phenotype of pathways in which the product of the variant gene participates, and so forth. Filtering processes can have inherent flaws and may contribute to false-negative results. This is especially true for dominant missense variants that have not been described in the literature, have been inherited from either parent (as opposed to occurring de novo in the proband), or have incomplete penetrance and/or variable expressivity.20 Another concern is the ability to interpret identified variants, especially missense and silent variants. Frameshift, nonsense, and canonical splice site variants, and deletion and duplication of several bases in regions within functional domains are considered likely pathogenic, although some The Journal of Molecular Diagnostics - jmd.amjpathol.org Laboratory Perspective Evidence-Based Targeted Gene Panels/WES/WGS Since the implementation of NGS in clinical laboratories, the focus has been on developing large (>50) gene panels.6,14,21 More recently the trend has shifted toward using WES and WGS. Although it is assumed that variants identified on gene panels are likely relevant to the patient’s phenotype, a finding not related to the primary clinical indication may still be identified. Such incidental findings are a certainty in WES and WGS. The chance of identifying incidental findings increases when the genes being tested increase in number and decrease in specificity with respect to the disease. For example, a highly focused gene panel test, consisting of only well-vetted genes for a narrow and specific phenotype (eg, five genes associated with Cornelia de Lange Syndrome), is unlikely to reveal information not directly related to the patient’s phenotype. However, when a large panel designed to evaluate patients who have a broad, or ill-defined phenotype (eg, ataxia and spasticity) is used, the chance of identifying an incidental finding is increased. For example, in the process of testing all genes known to cause ataxia, one might identify a heterozygous variant in the ATM gene, which confers a significantly increased risk of developing breast cancer. Both WES and WGS provide at least some information on 90% to 95% of all known genes, even though the purpose of doing the test is to identify the underlying cause of a more-or-less specific phenotype in a patient. Because all of the data must be filtered in a variety of ways to get to the molecular basis of the disease, many variants in many genes must be examined to obtain a definitive answer. It is very likely that heterozygosity (carrier status) for autosomal recessive or X-linked disorders will be uncovered, and there is also a chance of identifying genes that convey risk of developing a late-onset disorder, including disorders with devastating neurologic effects, or cancer. 109 Hegde et al Although WGS provides more data than WES, the risk of identifying incidental findings is not expected to be much higher because most of the additional data present in a WGS test is not currently interpretable with respect to human disease. From the laboratory’s perspective, the reporting of a specific set of secondary findings (as recommended by the ACMG) has several implications from patient consent, laboratory analysis/ workflow, and cost standpoints. It is generally understood that obtaining consent for testing is the responsibility of the ordering health care provider and not of the laboratory. If the laboratory has decided to provide an opt-out for patients, it will be necessary to modify the analysis to reveal or mask information on the specific genes such as those recommended for reporting by ACMG. As recommendations change, laboratories must implement workflow changes continuously. The review and interpretation of variants in the 56 recommended ACMG genes requires significant effort from the bioinformaticists/analysts, genetic counselors, and laboratory directors as they work to determine which variants are reportable and which do not meet criteria for reporting. In addition, each reported incidental finding should be confirmed by Sanger sequencing, incurring further expense and increasing turnaround times. The recommendation to report incidental findings in all persons who undergo WES is an additional burden on laboratories that perform full WES on parents and/or siblings only in the process of attempting to identify the proband’s disease-causing variant(s). To perform additional analyses on those persons (and interpret and confirm the findings) involves more effort and expense, and puts those laboratories at a disadvantage compared with laboratories that only perform targeted analysis of family member samples for segregation analysis of presumptive disease variants in the proband. Current limitations of NGS include major challenges in interrogating medically significant genes with highsequence homology. These include genes with complex sequence contexts such as pseudogenes, genetic rearrangements, or a high GC content. Exclusion from analysis is a possibility but clearly not optimal for genes of high medical relevance, especially those for which ACMG recommends returning results regardless of the patient’s clinical phenotype/indication for testing. Out of the 56 genes on the ACMG recommended list, at least eight have one or more pseudogenes associated with them (MYH7, MYLK, PKP2, PMS2, PTEN, SCN5A, SDHC, and SDHD). These regions should be sequenced with great attention, using more stringent criteria, to ensure reporting of variants only from the active copy of the gene and may require additional confirmatory testing. The Patient Perspective Decision Making and Insurance Considerations Studies by Tabor et al22,23 on the informed consent process in research WGS found that, although all family members involved wanted to receive results related to the condition for 110 which they were being tested, they differed on their desire to receive incidental findings, even among members of the same family. Several participants also felt that it was important to be able to change their preferences between the time of consent and the time of receiving results. A study of institutional review board perspectives on the return of genomic research results noted respondents were firm in their position that the research participants’ right to know/not know information about themselves be respected.24,25 The National Heart, Lung, and Blood Institute issued updated guidelines in 2010 on reporting genetic research results to study participants.26 They recommend that genetic results should be offered to study participants if the results meet certain criteria, including during the informed consent process or subsequently, the study participant has opted to receive his or her individual genetic results. An overriding regard for the right of the participant/ patient in deciding what results to receive is evident in these studies. Although these studies were conducted with persons undergoing WGS on a research basis, it is reasonable to expect similar results from clinical WES/WGS participants. There are a number of reasons that persons may be hesitant to receive incidental finding results. First, the expressivity of genetic variants known to be highly penetrant in high-risk communities is unknown for low-risk communities. In this vein, the US Preventive Services Task Force recommended against BRCA gene testing in low-risk populations (Risk Assessment, Genetic Counseling, and Genetic Testing for BRCA-Related Cancer in Women, Topic Page. US Preventive Services Task Force; http://www.uspreventiveservicestaskforce.org/Page/ Topic/recommendation-summary/brca-related-cancer-riskassessment-genetic-counseling-and-genetic-testing, last accessed February 9, 2015). Patients may not want information that may cause more distress than clinical benefit.8 Second, it is possible that incidental findings revealed by WES/WGS testing may prevent a person from obtaining life insurance, long-term care insurance, and/or disability insurance. Although the Genetic Information Nondiscrimination Act protects against health insurance and employment discrimination, it does not extend to these other areas of insurance. Patients should be informed of this possibility before testing. Patients should be given the opportunity to carefully consider the risks associated with possible incidental findings versus the possible risks of insurance discrimination and then decide for themselves whether they want to receive the incidental findings. Without an opt-out option, patients only have the choice of undergoing WES/WGS, which could potentially diagnose their condition and at the same time leave them unable to obtain life/ disability/long-term care insurance, or declining the opportunity for diagnosis to prevent insurance discrimination. Public Databasesefrom AMP Letter to Presidential Commission In 2013, AMP submitted public comments to the Presidential Commission for the Study of Bioethical Issues on jmd.amjpathol.org - The Journal of Molecular Diagnostics Reporting Incidental Findings in Genomics the topic of genetic and genomic testing. In that letter, the creation and maintenance of public databases for the curation of genomic variants was addressed. To reduce the number of variants of uncertain significance in both primary and incidental findings, information must be gathered on the possible clinical consequences of individual variants. As most genetic conditions are rare, information from any single health care provider’s patients will likely be of limited value. International databases of de-identified data will provide much more valuable information that can be used to interpret variants. Resources must be dedicated to the creation, development, and maintenance of centralized, curated databases of genomic variants, to which all laboratories should be strongly encouraged to submit de-identified data, including individual variants identified, relevant associated clinical information, and acquired information about potential pathogenicity to publicly available databases such as ClinVar database from the National Center for Biotechnology Information. Data must be submitted with appropriate safeguards to protect patient privacy. Resources should be provided to allow for continual curation and updating of the information contained within the database to ensure accurate and timely classification of variants. Comparison with Incidental Findings in Other Areas of Medicine Reporting of incidental findings is not a new problem in medicine. Incidental findings in radiology are common. A typical example would be a scan ordered to assess the kidneys, which necessarily includes the adrenal glands (present at the top of the kidneys).27 If a lesion is present in the adrenal gland, the radiologist cannot avoid seeing it even if the examination was undertaken only because of suspected kidney disease. Most such incidentalomas prove benign but can trigger expensive anxiety-inducing work-ups. Similarly, in the field of genetics incidental findings are not uncommon. For example, chromosome analysis of leukemia might find an unexpected complement of sex chromosomes, or evaluation of the segregation of a mutation in a family may reveal misattributed paternity. A feature common to all these examples is that the clinician, although not specifically looking for the incidental finding, cannot avoid the observation. The ACMG report, supported by some commenters, argues that incidental findings from WES/WGS are similar to incidental findings in other areas of medicine and must be reported, as in common practice.7,13 However, other commenters, as well as this document,28 point out that additional findings, beyond the genes analyzed to answer the clinical question that prompted testing, are not evident without significant extra effort directed toward that end.8,10 The use of the term incidental finding for both types of results is unfortunate, because the two types of findings are The Journal of Molecular Diagnostics - jmd.amjpathol.org fundamentally different, one being unavoidable and the other requiring additional analysis and interpretation. Genetic Counseling and Consenting Perspective The consenting process for WES and WGS is critically important and should be performed by a person highly trained in genetics such as a geneticist or genetic counselor. Because WES or WGS is much more complex than consenting to single-gene analysis for a Mendelian condition, patients and their families will need to be educated about the possible types of results that could be returned. The medical professional needs to be familiar with the testing protocol and consent form used by the laboratory, because this will affect the information the professional provides when obtaining consent from the patient/family.29 Medical professionals who order WES/WGS should also know that laboratory policies for analysis and reporting of incidental findings may change as they carefully consider advances in technology and the effect of implementing the ACMG recommended panel as well as the recommendations of other groups. Each laboratory should clearly indicate on their consent form if it routinely performs analysis for incidental findings such as the ACMG recommended additional 56 genes or whether it explores an even further expanded list of incidental findings unrelated to phenotype (such as pediatric-onset conditions, adult-onset conditions, carrier status for recessive conditions, or pharmacogenetic markers). Laboratories should also be as specific as possible in listing the genes to be analyzed and in defining the types of incidental findings they routinely report, especially if they deviate from the ACMG recommended list, because this will aid the genetics professional in obtaining the most accurate informed consent possible. If they exclude certain types of conditions from being reported as incidental findings (ie, adult-onset neurodegenerative conditions with no cure) that should be specified in the consent. The consent should clearly delineate whether the patient or other family members have the option of choosing not to receive incidental results. If several family members are undergoing exome sequencing to help aid in the variant analysis and diagnosis of the proband, each of these persons should give consent on a separate written document so that their independent desire to opt-in or opt-out of testing for incidental findings can be respected. They should also be informed about how they are to receive incidental findings if they are not the proband, because the results from the incidental findings in other family members should remain confidential. In addition, some laboratories that typically provide incidental findings may feel strongly about not violating the principle of a minor’s autonomy by not reporting incidental findings for adult-onset conditions for a patient who is younger than 18 years. They may decide not to test for adult-onset conditions in minors. In a joint policy statement and technical report, the ACMG and the American Academy of Pediatrics continue to support deferring the testing of late-onset conditions until adulthood.30 111 Hegde et al Table 1 Education Material in Genomics Target audience Ordering practitioners Concept to address Materials/resources testing laboratory may provide Clinical utility Scientific references Limitations of the technology Practice guidelines Scientific references Likelihood of incidental findings Scientific references Potential effect of incidental findings on the patient Example of potential scenarios Practitioner/patient discussion NA Genetic counseling partnership Recommended source for materials Comments General: manufacturer/ References should meet professional appropriate levels of evidencesocieties; testbased medicine specific: laboratory* Overview of potential for falsely positive/negative results. Specific genetic aberrations not identified (ie, large indels) Types: carrier status, inheritance pattern; particular attention should be paid to effect on patient and other family members Medical effect (management); psychological effect (anxiety/ relief); financial effect (life, disability insurance, etc) Provide adequate time and information for an informed decision Expertise available to the provider and/or patient before and after testing Find a genetic counselor through the National Society of Genetic Counselors (http://www. nsgc.org, last accessed December 1, 2014) Federal laws that govern genetic References to the Genetic Federal protections to patients about the use of genetic testing Information information for employment Nondiscrimination Act and health insurance and patient protection and Affordable Care Act Local and state laws that govern Specific local requirements may genetic testing affect consent forms, disclosure information, etc Uphold patient autonomy Explanation for the Patient empowerment for final necessity of excellent decision making communication skills Same as ordering practitioners See above Referring More detailed summary of Scientific references/ laboratorians/ Limitations of the technology limitations (ie, exon drop out) laboratory-specific pathologists and how the laboratory handles/ limitations (method reports such limitations based) Patients Uphold patient autonomy Educational packet; online materials for patients General consenting process to Educational packet for include an explanation of: patients General description of the test Purpose of the test Offer pretest genetic counseling Results may indicate a predisposition to a disease, further testing may be pursued General: manufacturer/ professional societies; testspecific: laboratory Laboratory Professional societies Professional societies NA Professional societies Professional societies/ laboratory Professional societies Laboratory Professional Societies Professional societies local/state requirements: laboratory Laboratory Referring physician Referring physician Physician (table continues) 112 jmd.amjpathol.org - The Journal of Molecular Diagnostics Reporting Incidental Findings in Genomics Table 1 Target audience (continued ) Materials/resources testing laboratory may provide Concept to address Incidental genomic findings, general discussion Variety of options for reporting incidental findings Comments Recommended source for materials Laboratory Patient has the right to choose Laboratory what information should be in the report (ie, targeted only vs targeted and incidental etc) Laboratory Level of certainty the findings predict a disease state Names/categories of persons and organizations to whom the results may be disclosed Identification of nonparental Particularly when parentrelationships proband triads are tested Support services before and after Referrals to appropriate Genetic counselors, medical testing professionals, as needed geneticists, medical specialists, therapists Laboratory Laboratory Laboratory *Laboratory provides the source of information to the referring physician who educates patient. NA, not applicable. Patients/families should be counseled on the numerous limitations of exome sequencing. First, in the context of the primary indication for the test, if a causative gene variant(s) is not identified for a disorder in question, a genetic etiology has not been excluded. They should understand that there are many genes not amenable to variant detection by using NGS technology. Next, should incidental findings be sought, not all genes or gene regions will be analyzable by NGS, so a negative result does not rule out a variant in the list of ACMG genes or any other expanded list of incidental findings. For example, the ACMG list includes the Lynch syndrome gene, PMS2.31,32 Since there are up to 16 pseudogenes for some exons in the PMS2 gene with a high level of extreme sequence similarity, it may not be possible to determine whether variants identified in the PMS2 gene are actually in the PMS2 gene or a pseudogene. Because of all these problematic genes, the recommendation from this workgroup is that pathogenic incidental findings should be confirmed by Sanger sequencing before reporting the result. In addition the common causative mutation in other ACMG genes may be a large deletion (eg, SMN1, which causes spinal muscular atrophy); large deletions are not amenable to detection by either NGS or Sanger sequencing and require other methodologies for detection. Laboratories should clearly describe on the consent document the type of variants reported for the primary indication, for example, pathogenic and likely pathogenic variants, variants of unknown significance identified in genes known to be associated with disease, and variants in genes of unknown function. They should describe how this differs from the types of variants reported for incidental findings, which should be restricted to prior reported known pathogenic variants and expected pathogenic variants that are of the type expected to be causative for the disorder. The The Journal of Molecular Diagnostics - jmd.amjpathol.org consent should clearly state the psychological risks and risks of insurance discrimination that could be the sequelae of reporting incidental findings. This is important because of the real difficulty persons who are carriers of cancer predisposition gene variants may face in getting life or disability insurance. Oversight and Regulation Laboratories must comply with federal and state regulatory requirements. At the federal level, the Clinical Laboratory Improvement Amendments (CLIA) regulations provide minimal standards for laboratory practice.33 The Centers for Medicare and Medicaid Services provide oversight and enforcement of the CLIA regulations. States such as New York and Washington have developed their own regulations comparable with federal oversight, and the Centers for Medicare and Medicaid Services has deemed these comparable with the federal requirements which allow these states to use their own oversight mechanisms (http://www.wadsworth.org/labcert/ clep/clep.html and http://www.hhs.gov/regulations, last accessed December 2, 2014). Some states impose additional requirements on laboratories beyond what is described in the CLIA regulations (http://www.healthit.gov/sites/default/ files/290-05-0015-state-law-access-report-1.pdf, last accessed December 2, 2014). For example, several states have regulations that address which person(s) is authorized to receive reports of clinical laboratory test results. A recently announced policy from the US Health and Human Services indicates that patients should be able to receive results directly from the testing laboratory. The US Food and Drug Administration primarily regulates manufacturers of tests that are sold to clinical laboratories for clinical testing (http://www.fda. 113 Hegde et al Table 2 Recommendations of the AMP Work Group on Incidental Findings 1. Test advantages and limitations should be made available to the physician and clearly stated on the laboratory reports. 2. Laboratories should develop a consent form, which clearly explains the test advantages and limitations, and the categories of variants reported (eg, diagnostic, carrier, pharmacogenetic markers). 3. Choice of opt-in or opt-out be given to patients and their families (in case of minors) in the precounseling session. 4. Laboratory clearly states which genes (ACMG-defined list, laboratory-defined list, or all genes) will be analyzed in incidental findings with the disclaimer that not all regions of all genes are always covered. 5. Only known pathogenic or likely pathogenic variants should be reported. 6. Reported pathogenic variants should be confirmed with an alternate technology. The method used for confirmation should be stated under the methodology section in the reports. 7. Laboratories should actively submit the list of pathogenic variants identified in their defined list of genes for which they choose to report incidental findings to public databases such as ClinVar, Human Gene Mutation Database, and Leiden Open Variation Database. ACMG, American College of Medical Genetics and Genomics; AMP, Association for Molecular Pathology. gov/MedicalDevices/ProductsandMedicalProcedures/InVitro Diagnostics/default.htm, last accessed December 2, 2014). To date, clinical sequencing tests are developed in the laboratory and do not come under the purview of the Food and Drug Administration. In 2013, the Food and Drug Administration cleared four NGS products, two cystic fibrosis tests and a platform in combination with a universal reagent kit. To date, the regulatory agencies described here have not addressed the issue of reporting incidental findings. Finally, the College of American Pathologists accredits medical laboratories in a voluntary system by using inspections according to checklists that are laboratory specific and regularly updated (http://www. cap.org/apps/cap.portal?_nfpbZtrue&_pageLabelZaccred itation, last accessed December 2, 2014). The Molecular Pathology Checklist (www.cap.org/molecularpathology checklist), updated July 31, 2012, states that laboratories should have a “policy regarding reporting of clinically significant genetic findings unrelated to the clinical purpose for testing” but does not specify what that policy should be. Thus, the laboratories are responsible for developing their own individual policies. CLIA requires that clinical laboratories establish the performance specifications of tests offered. These include accuracy, precision, analytic sensitivity and specificity, reference range, reportable range, and other relevant characteristics. This requirement applies equally to all reported findings of a clinical test with no distinction between test results that address the reason the test was ordered or are incidental. However, this standard does not require that all test results meet identical criteria, just that the performance specifications and limitations of the test are documented. In practical terms, if incidental findings are reported, the methods used must be validated with accepted protocols that meet regulatory standards and, if applicable, accreditation standards. It is clearly not permitted to report incidental findings if the laboratory has not established that the performance specifications of this aspect of testing meets these standards. At the present time, regulatory and accreditation requirements do not distinguish between primary and incidental findings. Therefore, assay development for both should be comparable, and the reporting of an incidental finding should be as reliable as the reporting of a primary test result. For example, the validation protocols should be comparable. In addition, the direct costs of validating, identifying, interpreting, and reporting incidental findings may also be significant, and it is not clear to what extent payers will reimburse for aspects of testing that are not explicitly ordered. 114 jmd.amjpathol.org The Need for Education in Genomics As genomic medicine increases in clinical utility and enters the everyday vernacular of the medical community, there is a tremendous need to ensure that the appropriate information and education reach the correct audiences. The education needs to be tailored to all of the stakeholder groups influenced by genomics. Stakeholders will likely include multidisciplinary care teams who can appropriately manage patients in an era of precision medicine. These groups include i) ordering practitioners (physicians, physician assistants, nurses, genetic counselors), ii) referring laboratorians and/or pathologists (eg, molecular laboratory geneticists certified by the American Board of Medical Genetics and Genomics), iii) patients (and family members, when applicable), iv) policy makers, and v) payers. In each of the groups listed in the previous paragraph, there clearly will be a wide range of educational levels, socioeconomic backgrounds, and abilities to access supportive services. Thus, a one-size-fits-all approach to education is wholly insufficient to deal with the particular questions and concerns of each stakeholder population. The importance of the education process in genomic testing and the possibility of incidental findings cannot be overstated. All stakeholders should be comfortable with the intent, likelihood, and potential effect of incidental findings before ordering any genomic evaluation. With the large amount of educational materials that should be provided to the variety of stakeholders, it is important to address potential sources for these materials (Table 1). Some materials, such as those directly related to a particular test, should be provided by the performing laboratory. For laboratory-developed procedures, there may not be any other resource accurately describing the test characteristics. Testspecific educational information should be a component of - The Journal of Molecular Diagnostics Reporting Incidental Findings in Genomics the informed consent process; in fact, some states (ie, New York) have civil rights laws that mandate this information be provided at the time of consent. For more general information, such as the limitations of a particular method, the molecular diagnostic community could look to the manufacturers and/or professional societies for the development of educational materials. Clearly, a robust education program should be developed in conjunction with professional societies. Workgroup Conclusion on Reporting of Incidental Findings The detection of genetic variants that may be of clinical significance but are unrelated to the indication for testing may result from NGS analysis of gene panels, whole exomes, and genomes. A great deal of controversy exists about whether these variants should be reported to the ordering physicians and the patients. The ACMG Working Group on Incidental Findings initially recommended that known pathogenic and predicted to be pathogenic mutations in a set of 56 genes be reported without regard to the age of the patient or the initial reason for testing, except for fetal samples. After extensive feedback, ACMG issued a clarification, stating that patients should be given an opportunity to opt-out of receiving incidental findings. This workgroup is of the opinion that each laboratory that provides NGS services must develop a policy for analysis and reporting of known and predicted pathogenic variants from the list of genes recommended by ACMG and other known and predicted pathogenic variants (Table 2). The laboratories’ policy should be based on its ability to provide this information with sufficient accuracy. We also support the practice that persons undergoing WES or WGS should have the opportunity to opt-in or opt-out of receiving a report that documents variants that are not relevant to the initial reason for testing. Each laboratory must also determine what information it is willing to provide to parents or other third parties, and whether these third parties will have the opportunity to opt-in or optout. This decision should be part of the patient education and informed consent process. Most patients will likely opt-in, given the opportunity to receive additional information that could be used to improve their health. However, health care professionals must be sensitive to the possibility that patients dealing with a difficult medical issue that led to the need for WES or WGS may not wish to have an additional burden of concerns placed on them about future health issues. In addition, the needs of specific cultural and ethnic groups must be accommodated. Every laboratory that performs NGS testing for clinical applications should be aware of the ethical and patient management issues associated with incidental findings. Laboratories should have policies in place that clearly define their approach to reporting the ACMG gene list and other incidental findings. NGS technology is evolving rapidly, and the rate of confirmation of single nucleotide variants is very high, but The Journal of Molecular Diagnostics - jmd.amjpathol.org the technology is not accurate for detecting copy number variants. It is likely that once a pathogenic variant is reported, additional family members will obtain targeted testing for themselves or prenatal diagnosis. Targeted testing will likely be performed with alternate method such as Sanger sequencing. For these reasons, this workgroup recommends that reported variants identified by NGS be confirmed with an alternate method. As the NGS technology evolves, it is likely that these recommendations will change. Only the known pathogenic and expected pathogenic variants should be included in the incidental finding report if the laboratory chooses to report these. The interpretation of variant pathogenicity should follow the ACMG Recommendation for Interpretation of Sequence Variants.19 The determination of pathogenicity for known variants is based on scientific literature, variant databases, and clinically validated/curated locus-specific databases, which support the assertion that the variants in question are causative of disease or not. For variant(s) that have not been reported previously, the known mechanism of disease and the ACMG guidelines should be followed. All of the known pathogenic and expected pathogenic variant(s) should be reported in accordance with the nomenclature recommended by the Human Genome Variation Society (http://www.hgvs. org/mutnomen, last accessed December 2, 2014). The report should include relevant references to the literature and references to variant databases to support the classification of the pathogenicity of the variant. Incidental findings should be clearly labeled as such and included in the full report sent to the ordering physician. One ongoing consideration is that often the ordering physician is not experienced in interpretation of reports that involve gene variants and their implications for disease. It will be very useful to include information in the report about the variant’s association with disease and to provide recommendations for clinical follow-up. The report should be written so that it can be understood without the need for a high level of genetic knowledge and should avoid the use of scientific jargon. The report should clearly outline the limitations of the analysis. For example, some genes, or parts of genes, may not be adequately sequenced (some region of the genes could have low or absent sequencing coverage), and some variants that are discovered may not be clearly interpretable for risk of clinical disease. This is important because the ordering physician and the patient might believe that the lack of reporting of an incidental finding equates with a lack of pathogenic variants. This workgroup appreciates that the field of genomic/ precision medicine continues to evolve rapidly and the clinical, laboratory, and ethical issues surrounding reporting of incidental findings needs continued evaluation from the multidisciplinary clinical community. It is likely that recommendations from professional societies will evolve as NGS technology matures, genomic education improves, and 115 Hegde et al regulatory authorities and other groups refine the standards for laboratory medicine with respect to genomic analysis. Disclaimer The AMP Clinical Practice Guidelines and Reports are developed to be of assistance to laboratory and other health care professionals by providing guidance and recommendations for particular areas of practice. The Guidelines or Report should not be considered inclusive of all proper approaches or methods, or exclusive of others. The Guidelines or Report cannot guarantee any specific outcome, nor do they establish a standard of care. The Guidelines or Report are not intended to dictate the treatment of a particular patient. Treatment decisions must be made based on the independent judgment of health care providers and each patient’s individual circumstances. AMP makes no warranty, expressed or implied, regarding the Guidelines or Report and specifically excludes any warranties of merchantability and fitness for a particular use or purpose. AMP shall not be liable for direct, indirect, special, incidental, or consequential damages related to the use of the information contained herein. References 1. Grody WW, Thompson BH, Hudgins L: Whole-exome/genome sequencing and genomics. Pediatrics 2013, 132:S211eS215 2. Kohane IS, Masys DR, Altman RB: The incidentalome: a threat to genomic medicine. 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