Clinical testing of BRCA1 and BRCA2: A worldwide snapshot of technological practices

Abstract

Clinical testing of BRCA1 and BRCA2 began over 20 years ago. With the expiration and overturning of the BRCA patents, limitations on which laboratories could offer commercial testing were lifted. These legal changes occurred approximately the same time as the widespread adoption of massively parallel sequencing (MPS) technologies. Little is known about how these changes impacted laboratory practices for detecting genetic alterations in hereditary breast and ovarian cancer genes. Therefore, we sought to examine current laboratory genetic testing practices for BRCA1/BRCA2. We employed an online survey of 65 questions covering four areas: laboratory characteristics, details on technological methods, variant classification, and client-support information. Eight United States (US) laboratories and 78 non-US laboratories completed the survey. Most laboratories (93%; 80/86) used MPS platforms to identify variants. Laboratories differed widely on: (1) technologies used for large rearrangement detection; (2) criteria for minimum read depths; (3) non-coding regions sequenced; (4) variant classification criteria and approaches; (5) testing volume ranging from 2 to 2.5 × 10⁵ tests annually; and (6) deposition of variants into public databases. These data may be useful for national and international agencies to set recommendations for quality standards for BRCA1/BRCA2 clinical testing. These standards could also be applied to testing of other disease genes.

Full text

ARTICLE OPEN
Clinical testing of BRCA1 and BRCA2: a worldwide snapshot
of technological practices
Amanda Ewart Toland
1
, Andrea Forman
2
, Fergus J. Couch
3
, Julie O. Culver
4
, Diana M. Eccles
5
, William D. Foulkes
6
,
Frans B. L. Hogervorst
7
, Claude Houdayer
8
, Ephrat Levy-Lahad
9
, Alvaro N. Monteiro
10
, Susan L. Neuhausen
11
, Sharon E. Plon
12
,
Shyam K. Sharan
13
, Amanda B. Spurdle
14
, Csilla Szabo
15
and Lawrence C. Brody
15
on behalf of the BIC Steering Committee
Clinical testing of BRCA1 and BRCA2 began over 20 years ago. With the expiration and overturning of the BRCA patents, limitations
on which laboratories could offer commercial testing were lifted. These legal changes occurred approximately the same time as the
widespread adoption of massively parallel sequencing (MPS) technologies. Little is known about how these changes impacted
laboratory practices for detecting genetic alterations in hereditary breast and ovarian cancer genes. Therefore, we sought to
examine current laboratory genetic testing practices for BRCA1/BRCA2. We employed an online survey of 65 questions covering four
areas: laboratory characteristics, details on technological methods, variant classification, and client-support information. Eight
United States (US) laboratories and 78 non-US laboratories completed the survey. Most laboratories (93%; 80/86) used MPS
platforms to identify variants. Laboratories differed widely on: (1) technologies used for large rearrangement detection; (2) criteria
for minimum read depths; (3) non-coding regions sequenced; (4) variant classification criteria and approaches; (5) testing volume
ranging from 2 to 2.5 × 10
5
tests annually; and (6) deposition of variants into public databases. These data may be useful for
national and international agencies to set recommendations for quality standards for BRCA1/BRCA2 clinical testing. These standards
could also be applied to testing of other disease genes.
npj Genomic Medicine (2018) 3:7 ; doi:10.1038/s41525-018-0046-7
INTRODUCTION
Clinical genetic testing of BRCA1 and BRCA2 began in the mid-
1990s, but was mainly limited to one laboratory in the United
States (US) and a small number of laboratories in Australia and
Europe. Twenty-five years later the number and types of patients
being offered BRCA1/BRCA2 testing has changed dramatically due
in part to changes in patent laws and increased recognition of
potential benefits of testing. Additionally, advances in high-
throughput sequencing technology have enabled laboratories to
offer tests that cover more genes. The testing panels are less
expensive than older tests and feature shorter turn-around times
(TAT). These changes in access and technology have led to a
similarly intense increase over the last 10 years in the number of
laboratories offering clinical genetic testing of BRCA1/BRCA2
specifically, multi-gene cancer gene panels for germline and
somatic variant analysis, as well as companies that offer whole-
exome and whole-genome studies that capture mutation
information on these genes. This has led to a diverse array of
options from which cancer genetics care providers can choose.
In 2013, a survey of 13 US laboratories offering BRCA1/BRCA2
sequence analysis revealed a large number of differences in
technology, gene coverage, analytic sensitivities, ability to detect
large rearrangements, cost, single-site analyses, and frequency of
reporting variant of uncertain significance (VUS).
1
Since 2013,
many additional laboratories now offer BRCA1/BRCA2 testing,
including laboratories that perform somatic mutation analyses.
Given the expanded testing and challenges faced by clinicians and
patients interested in comparing test offerings, the Breast Cancer
Information Core (BIC) Steering Committee developed and
administered a survey aimed to address the question of variation
in clinical laboratory practices for BRCA1/BRCA2 testing around the
world. The BIC is an open access, online database, which began in
1995, to catalog sequence variants in BRCA1/BRCA2.
2
The goal of
this study was to obtain a snapshot of current genetic testing
laboratory practices for BRCA1/BRCA2 worldwide. We believe this
study highlights similarities and differences between laboratories
including technologies utilized, regions of the genes commonly
assessed, as well as other quality control metrics employed. These
findings help us to understand international differences in testing
protocols and standards.
Received: 21 November 2017 Revised: 11 January 2018 Accepted: 16 January 2018
1
Departments of Cancer Biology and Genetics and Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA;
2
Fox Chase Cancer
Center, Philadelphia, PA 19111, USA;
3
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA;
4
USC Norris Comprehensive Cancer Center,
University of Southern California, Los Angeles, CA 90033, USA;
5
Faculty of Medicine University of Southampton, Southampton S016 5YA, UK;
6
Departments of Human Genetics,
Medicine and Oncology, McGill University, Montreal QC, CanadaH4A 3J1;
7
Family Cancer Clinic, Netherlands Cancer Institute, Amsterdam 1006 BE, Netherlands;
8
Oncogenetics
and INSERM U830, Institut Curie, Paris and Paris Descartes University, Paris 75248, France;
9
Faculty of Medicine, Shaare Zedek Medical Center, Hebrew University of Jerusalem and
Medical Genetics Institute Jerusalem 9103102, Israel;
10
Department of Cancer Epidemiology, Moffitt Cancer Center, Tampa, FL 33612, USA;
11
Department of Population Sciences,
Beckman Research Institute of City of Hope, Duarte, CA 91010, USA;
12
Baylor College of Medicine, Houston, TX 77030, USA;
13
Mouse Cancer Genetics Program, Center for Cancer
Biology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702-1201, USA;
14
Genetics and Computational Biology Division, QIMR Berghofer Medical
Research Institute, Herston, Brisbane QLD QLD 4006, Australia and
15
National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence: Amanda Ewart Toland (Amanda.toland@osumc.edu)
www.nature.com/npjgenmed
Published in partnership with the Center of Excellence in Genomic Medicine Research

RESULTS
Laboratory locations
Survey links were sent to BRCA1/BRCA2 genetic testing labora-
tories around the world. Every continent, except Antarctica, was
represented by at least one testing laboratory (Fig. 1a). The bulk of
testing laboratories responding to the survey were from Europe
which reflects the demographics of the laboratories which were
sent the survey invitation (Fig. 1b). Eight US laboratories
completed the survey (Fig. 1c).
Technologies used
Massively parallel sequencing (MPS) was the most common
method utilized by non-US laboratories to identify BRCA1/BRCA2
variants with the most common platforms being Illumina MiSeq
(59%; 46 of 78 laboratories) and Ion Torrent (22%, 17 of 78
laboratories) (Fig. 2a). Of the 78 non-US laboratories responding,
six reported Sanger sequencing as their only method of
sequencing (8%) and 27 additional laboratories (35%) used Sanger
as at least one modality, mostly to confirm variants identified by
other methods. Twenty-six of 33 (79%) non-US laboratories
reporting on both technology used and use of gene-panels
include ten or more genes on their panel; seven laboratories
reported using MPS for BRCA1/2 only. Many laboratories (31/78;
40%) reported using multiple technologies sequentially for initial
discovery. There was less variability in the approach for large
rearrangement analysis with 86% of non-US laboratories (66 of 77)
utilizing multiplex ligation-dependent probe amplification (MLPA);
however, 31% (24 of 77) also used data from a MPS platform,
some noting that they used MLPA analyses to confirm rearrange-
ments identified by MPS (Fig. 2b). Five of 77 laboratories (6%) did
no duplication/deletion analyses, and one reported sending DNA
to an external laboratory if no variants were detected by MPS. All
eight US-laboratories used MPS technologies as one modality for
sequence variant detection, and six reported using more than one
technology (Fig. 2c). All US-laboratories responding offer gene
panels and have panels available that include more than ten
genes. Five of the eight US-laboratories also reported using Sanger
sequencing, mostly for confirmation of variants found through
other methods. Only half of the US laboratories used MLPA which
differs from the non-US laboratories (66 of 77; 86%) (Fig. 2d).
Seven of the US laboratories reported using more than one
platform to identify large sequence variants; six used chromoso-
mal microarray analysis or array comparative genomic hybridiza-
tion (aCGH) (75%) in combination with MPS (five of eight; 63%).
One US laboratory reported solely using MPS for detection of large
sequence events.
The survey did not specifically ask testing laboratories whether
they only performed panel analyses, only if they performed single
gene analyses or did both types of tests. However, laboratories
indirectly answered this question when addressing TAT. All eight
US laboratories responding to the survey offered panel-testing of
multiple cancer susceptibility genes. Of the eight, only one does
not offer single gene analysis. For the 47 non-US laboratories, 40
(85%) noted that they perform multi-gene panels; two did not
respond to the BRCA1/BRCA2 only TAT question. Forty-five
laboratories reported TAT for BRCA1/BRCA2 testing of which eight
do not perform panel testing (18%).
Regions of BRCA1/BRCA2 sequenced
For BRCA1/BRCA2 analyses, coding exons were covered in full by
all laboratories but only six of 70 non-US laboratories total (9%)
and no US laboratories reported doing full intronic sequencing. Of
the 54 non-US laboratories reporting the length of intronic
sequence included in their tests, 30 (56%) sequenced 11–20 bp of
intronic sequence, 12 (22%) sequenced 6–10 bp of intronic
sequence and two laboratories sequenced up to 5 bp at intron/
exon junctions (Fig. 3a). Twelve non-US laboratories responded
with other answers including four laboratories that performed
complete intronic sequencing. Twenty-three of 54 non-US
laboratories (43%) sequenced non-intronic non-coding regions
of BRCA1/BRCA2 including promoters, enhancers, 3′untranslated
regions (UTRs), and 5′UTRs (Fig. 3b). US laboratories sequenced
similar intronic regions as non-US laboratories with three
sequencing 11–20 bp (43%), one sequencing 6–10 bp (14%), one
sequencing 20 bp proximal to the 5′end of an exon and 10 bp
distal to the 3′end of an exon (14%), one sequencing up to 5 bp of
introns plus all previously established intronic variants (14%) and
one sequencing all previously established intronic variants. Six of
seven US laboratories reported sequencing additional non-
intronic, regulatory regions of BRCA1/BRCA2. One laboratory noted
that they reported variants in established clinically relevant non-
coding regions. We did not ask specifically whether the
laboratories reported sequence variants from these regions.
Variant confirmation
If a sequence variant was identified, over half of non-US (56%; 29
of 52) and US laboratories (71%, five of seven) confirmed all
variants of uncertain significance (VUS), pathogenic and likely-
pathogenic sequence variants by another method. Twenty-seven
percent of non-US laboratories and 29% of US laboratories
confirmed only pathogenic or likely pathogenic variants. Sanger
sequencing was the most common method to validate variants
(98%; 50 of 51 non-US laboratories and 86%, six of seven US
laboratories), although 43% of US laboratories (three of seven)
began confirmation by repeating analysis with the original
technology. MLPA was used to confirm duplication or deletion
variants in 61% of non-US laboratories (31/51) and 57% of US
laboratories (3/7). Other methods used to confirm large rearrange-
ments by US laboratories included aCGH, quantitative PCR (qPCR),
and the PacBio system.
Coverage of Sequencing
There was a large range in both average read depth and minimum
read depth required to meet quality assurances for MPS (Table 1).
Of the 37 non-US laboratories reporting read-depths, the median
read depth across all genes on their cancer gene platforms was
476 with an average read depth of 759. These values were lower
for BRCA1/BRCA2 with a median read depth of 100 and an average
read depth of 484. US laboratories reported a median read depth
across all MPS platforms of 738 with a median of 425 and a higher
read-depth for BRCA1/BRCA2 (average = 820, median = 500). Forty-
two non-US laboratories reported a minimum read depth required
for BRCA1/BRCA2 analysis; these ranged from 20 to 500 reads for
BRCA1/BRCA2. When an exon failed to meet the minimum quality
standards for that laboratory, most non-US laboratories used
Sanger sequencing for the failed region (67%, 31 of 46) or a
combination of repeating the entire or part of the assay and/or
Sanger sequencing the affected region (22% 10 of 46). A small
number of non-US laboratories repeated the entire assay (4%; 2 of
46) or repeated the assay around the affected region (2.1%, 1 of
46) without any Sanger sequencing. In contrast, four of the US
laboratories (50%) repeated the entire assay and/or performed
Sanger sequencing of the affected region.
Analytics-sensitivity/specificity
Forty-six non-US laboratories answered questions related to the
analytical sensitivity of their BRCA1/BRCA2 testing. Of these, 16
(30%) had not calculated an analytic sensitivity for identification of
known BRCA variants. Twenty-six non-US laboratories (52%)
provided a value with an average analytical sensitivity of 98.3
and a median of 99. All eight US laboratories answered questions
related to sensitivity and provided an estimated analytical
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AE Toland et al.
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1234567890():,;

Fig. 1 Geographical location of participating BRCA1/BRCA2 testing laboratories. The geographical location of participating laboratories is
shown as pins on the world map for non-US laboratories (a), European laboratories (b), and US laboratories (c). Only laboratories that
completed at least half of the survey questions are shown. Two of the US laboratories have overlapping pins as they are located in the San
Francisco Bay Area. OpenStreetMap and ZeeMaps hold the copyright for the maps
Clinical testing of BRCA1 and BRCA2
AE Toland et al.
3
Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2018) 7

sensitivity for detection of sequence variants with an average of
99.6% and a range of 98–100%. Seven of eight US laboratories
reported the number of samples used to calculate the sensitivity
with an average of 598, a median of 500, and a range of 20–1864.
The laboratories were also asked questions about the sensitivity,
false discovery rate (FDR), and positive predictive value (PPV) of
their method for identifying insertion/deletions (indels). Of the 24
non-US laboratories responding, 21% (5/24) did not know this
information. Of the 16 laboratories that reported sensitivity as a
value, the average was 97% and the median was 99.5% with a
range of 85.2 to 100%. Four US laboratories reported a FDR with
an average of 2.25%, a median of 2.5% and a range of 0–4.
Multiple laboratories noted that FDR and PPV rates were difficult
to determine. Of the 20 non-US laboratories responding with
values, the average reliably detected indel size was 31 bp with a
median of 21 bp and a range of 1–104 bp. One non-US laboratory,
using Sanger sequencing, noted that they could reliably detect
indels of less than 100 bp. For the five US laboratories responding,
one noted that sensitivity, FDR, and PPV for indels were not
calculated. For the other four laboratories, three noted a sensitivity
of 100% with 95% confidence intervals of above 99.9% and a
specificity of 100% and/or a FDR of 0%. Six US laboratories
reported the size of reliably detected indels. Reliable detection
varied between deletions and duplications; deletions of an
average size of 33 bp with a median of 30 bp and a range of
20–40 bp and duplications of an average length of 23 bp, a
median of 25 bp and a range of 10 to 40 bp were considered
optimal for detection.
Variant classification
A number of variant interpretation guidelines were used. All
reporting laboratories responded that variant interpretation was
performed by in-house staff. For non-US laboratories, the majority
had a board certified medical geneticist or molecular geneticist on
their interpretation team (74%, 35/47). The remaining laboratories
had genetic counselors, individuals with clinical genetics expertise
related to the specific gene or an expert panel performed variant
classification. Almost half of non-US laboratories (47%, 22 of 47)
and 38% of US laboratories (3/8) reported using American College
of Medical Genetics and Genomics (ACMG) guidelines for variant
interpretation.
3
The remaining laboratories reported using in-
house guidelines (non-US laboratories 28% 13 of 47; US
laboratories 63%) or country or organization-specific guidelines
(non-US laboratories 21%). However, in the text descriptions,
many of the laboratories using “in house”guidelines relied at least
in part on guidelines from expert agencies. Guidelines or reference
databases used to aid in classification included Evidence-based
Network for the Interpretation of Germline Mutant Alleles
(ENIGMA) (enigmaconsortium.org/library/general-documents),
Vereniging Klinisch Genetische Laboratoriumdiagnostiek (www.
vkgl.nl/nl), Association for Clinical Genomic Science (www.acgs.uk.
com), and others.
4
Laboratories described a mixture of different
resources and databases used to help in classification including
ClinVar, literature searches, Alamut Visual (www.interactive-
biosoftware.com/alamut-visual), Sorting Intolerant From Tolerant
(SIFT)(sift.jcvi.org), Polyphen2 (genetics.bwh.harvard.edu/pph2),
dbSNP (www.ncbi.nlm.nih.gov/projects/SNP), Breast Information
Core Database (BIC)(research.nhgri.nih.gov/bic), Leiden Open
Fig. 2 Methods used for BRCA1/BRCA2 variant identification. The number and percentage of non-US (a/b) and US laboratories (c/d) reporting
use of each method for identification of BRCA1/BRCA2 sequence variants (a/c) and large rearrangements (b/c) are noted. The percentage of
laboratories reporting the use of more than one technology for variant detection was a40%, b38%, c75%, and d75%. Multiplex ligation-
dependent probe amplification (MPLA); massively parallel sequencing (MPS); array comparative genomic hybridization (array CGH)
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AE Toland et al.
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npj Genomic Medicine (2018) 7 Published in partnership with the Center of Excellence in Genomic Medicine Research

Variation Database (LOVD)(www.lovd.nl/3.0/home), Universal
Mutation Database (UMD)(www.umd.be/BRCA1), and BRCA
Exchange (brcaexchange.org).
5–9
Interpretation of VUS is an important clinical issue. Interestingly,
more than half (52%, 24 of 46) of the responding non-US
laboratories had not specifically calculated their BRCA1/BRCA2 VUS
rates. Of those that determined their rate of BRCA1/BRCA2 VUS,
these varied widely from 3–50% with an average and median VUS
rate for BRCA1 of 14 and 13% and for BRCA2 of 16 and 13%. Some
caution should be taken for interpretation of these figures as the
definition of VUS (e.g., all VUS versus VUS per individual) and
thresholds for calling vary between laboratories; also some
laboratories may have reported all sequence variants and not
just VUS (Supplemental Table 1). For the five US laboratories that
calculated BRCA1/BRCA2 VUS frequencies, the percentage of
individuals/tests with a VUS ranged from less than 2 to 6%.
Segregation analyses can be helpful for classifying variants.
Seventy percent (33 of 47) of non-US laboratories offered
variant-specific testing for family members when a VUS was
identified and another 15% of laboratories (7 of 47) offered VUS
testing depending on the circumstance. All US laboratories offered
VUS-family studies, but testing was limited to defined circum-
stances (Supplementary Table 2).
As VUS can be reclassified as additional data become available,
we asked how often laboratories reclassified VUS. Most non-US
laboratories (57%, 27 of 47) specified that reclassification was
done on an ad hoc basis, 9% (4 of 47) re-assessed VUS at least
annually, and 23% of laboratories (11 of 47) re-assessed VUS every
1–3 years. Three non-US laboratories did not reassess variants
(6%). This is in contrast to US laboratories in which 57% (four of
seven) reassessed variants less than once a year, 29% (two of
seven) reclassified variants on an ad hoc basis and one laboratory
reassessed VUS every 1–3 years. One US laboratory reported that
VUS were assessed daily due to automated reclassification tools
used in real time. When a VUS was reclassified, regardless of
classification, all eight US laboratories and 39% (18/46) of non-US
laboratories recontacted the ordering provider. Thirty-three
percent (15/46) of non-US laboratories recontacted the ordering
provider only when the VUS had been reclassified as pathogenic
or likely pathogenic. The remaining non-US laboratories did not
automatically contact the ordering physician (11%, 5 of 46) or only
did so when asked by the ordering provider (13%, 6 of 46).
To aid in classifying missense and other non-truncating variants
as pathogenic or not, it is useful to know how frequently a variant
has been observed in populations being tested. One third of non-
US laboratories (36%, 16 of 45) did not report any variants to any
databases, one third (33%, 15 of 45) shared BRCA1/BRCA2 data
with multiple open-access databases, and the remainder of labs
reported to only one database (22%, 10 of 45) or to private or
member-only databases (9%, 4/45). Of laboratories that reported
variants the most common databases were LOVD (40%, 18 of 45),
BIC (36%, 16 of 44) and ClinVar (27%, 12 of 45). US laboratories
were more likely to submit sequence data to public databases.
Seven of eight US laboratories (88%) reported sequence variants
to ClinVar; one laboratory did not report to public or private
databases.
Testing volume
Variant interpretation, TAT, and some quality measures may be
impacted by the testing volume. There was significant variation
among laboratories responding to this survey question. Forty-
three non-US laboratories described the number of BRCA1/BRCA2
tests performed between October 2015 and September 2016; the
number of tests ranged from 2 to 2025 with an average number
per year of 568 and a median of 300. Twelve laboratories
performed fewer than 100 tests per year. Only three US
laboratories answered the question on the number of tests
performed in the year from October 2015 to September 2016. The
range was ~45,000 to 252,223 which surpassed all non-US
laboratories by an order of magnitude (maximum of 2025); this
may be influenced by population size of country, the length of
time that testing has been available to clinicians, country-specific
guidelines for testing, and differences in marketing practices.
Client-related concerns
When there is a choice of laboratory and the patient and primary
care provider are using BRCA1/BRCA2 testing results to aid in
surgical decision making, TAT is an important consideration. Of 38
non-US laboratories reporting, there was an average TAT for gene-
panel testing of 6.5 weeks with a median of 5.6 weeks and a range
of 11 days to 6 months. For BRCA1/BRCA2 single-gene or small
panel analysis specifically, there was an average TAT of 4.9 weeks
(median = 4.0 weeks, range 0.5–4 months) for the 45 laboratories
responding. This contrasts with US laboratories that had a much
shorter average TAT of 2.4 weeks for gene-panels of over ten
genes with a median of 2.6 weeks and a range of 1.1 to 3.5 weeks.
For single gene analysis or small panels (six US laboratories
reporting), there was an average TAT of 1.7 weeks with a median
of 1.5 weeks and a range of 6.5 days to 2 weeks. One US laboratory
Fig. 3 Non-coding regions assessed. Of 54 non-US laboratories reporting, the number that sequenced intronic regions (a) or non-coding non-
intronic regions (b) are shown for different categories. Most of the intronic sequence refers to sequence near intron/exon boundaries. *Only
sequencing previously established clinical relevant intronic variants. Other for introns includes multiple categories of size of introns and other
for non-intronic regions includes “non-specified”, partial or only intronic non-coding regions. Twenty-seven laboratories answered the non-
intronic regulatory regions of BRCA1/BRCA2 question
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Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2018) 7

noted that there were additional options to expedite test results if
requested. To determine if TAT was influenced by the number of
tests performed per year, we plotted the TAT for BRCA1/BRCA2
tests in days by number of tests performed per year for the three
US laboratories and 38 non-US laboratories for which both values
were present (Fig. 4). There was no correlation between TAT and
testing volume (Pearson’s correlation all labs combined = −0.28;
non-US labs only was 0.02). There was also no correlation between
dedicated staff and TAT (Pearson’s correlation for all labs = −0.14;
non-US labs = 0.009). There was a negative correlation for US
laboratories with decreased TAT associated with increased
numbers of staff and increased testing volume, but these were
based on only three US laboratories (Pearson’s correlation US labs
=−0.72 and −0.76, respectively).
Laboratories were asked whether there were national or
regional guidelines to determine when to order a test and which
genes to include on clinical tests for hereditary cancer syndromes
(Supplemental Table 1 and 2). For the seven US laboratories that
responded, three (43%) depended on the referring physician to
determine appropriateness, two (29%) used National Comprehen-
sive Cancer Network and/or payer/insurance guidelines and one
(14%) used insurance carrier guidelines. For non-US laboratories,
almost half (20 of 41 laboratories) depended on the ordering
physician to determine the test being ordered and the other half
(21 of 41) used specific guidelines. The specific guidelines for
inclusion of a gene on a clinical panel for non-US laboratories
varied: 12 of 41 laboratories (29%) had no country-specific
guidelines, eight (20%) had defined genes that were covered by
insurance, and 20 laboratories (49%) had regional or national level
guidelines.
DISCUSSION
To our knowledge, this is the first international survey of
laboratories performing clinical testing of BRCA1/BRCA2 since the
adaptation of MPS. MPS use for clinical testing has been predicted
to introduce challenges in workflow, interpretation, and result
reporting.
10
By sampling the variety of technological approaches
used by testing laboratories and highlighting similarities and
differences, our goal is to provide information for testing
laboratories and agencies providing guidelines for quality
laboratory practices.
Across the world, MPS platforms were the preferred method for
detection of non-rearrangement BRCA1/BRCA2 sequence variants.
All laboratories interrogated all coding BRCA1/BRCA2 exons with
varying lengths of introns sequenced. Although many survey
responses were similar between non-US and US laboratories, our
survey revealed some key differences. Non-US laboratories were
more likely to use MLPA as one modality for large rearrangement
detection (86%) compared to US laboratories (50%) who were
more likely to use aCGH (75%) and MPS (88%). Most US and non-
US laboratories utilized MPS as the prime modality for variant
detection, but perhaps due to testing volume, more non-US
laboratories used Illumina MiSeq (59%) instead of the higher
capacity Illumina HiSeq used by most US laboratories (63%). US
laboratories were much more likely to use in-house methods of
classifying VUS (63%) compared to non-US laboratories (26%).
Non-US laboratories were more likely to use country-specificor
expert panel-specificBRCA1/BRCA2 guidelines (21%); although
three of eight US laboratories used ACMG or modified ACMG
guidelines.
3
US laboratories were more likely to share sequence
results with public databases (88% reported to ClinVar) than non-
US laboratories (55% reported to at least one public database).
Importantly, over one third of non-US laboratories did not report
variants to any databases or only reported to private/member only
sites (9%). From a client point of view, US laboratories have a
substantially shorter average TAT for BRCA1/BRCA2 or small gene
panels. This difference may be driven by competition between
companies in the US, the distribution of commercial versus
academic laboratories, and/or differences in insurance coverage
versus national health care coverage for testing. Finally, respond-
ing US laboratories performed many more BRCA1/BRCA2 tests
(lowest reported 45,000) per year relative to non-US laboratories
(range 1–2025), although only a small number of US laboratories
answered this question. The vast difference in testing volume
between US and non-US laboratories may be due in part to
differences in the population size of responding countries or in
Table 1. Massively parallel sequencing average and minimum read depths
Number of reads non-US laboratories Number of reads US laboratories
BRCA1/BRCA2 specifically
a
All genes on panel
b
BRCA1/BRCA2
c
specifically All genes on panel
c
Average read depth (median read depth) 483.5 (101) 758 (476) 820 (500) 738.3 (425)
Range of read depth 40–7000 40–7000 250–2400 150–2400
Average minimum read depth (median read
depth)
86.6 (50) 77.7 (40) 37.8 (50)
d
33.5 (50)
d
Range of minimum read depth 10–500 10–500 15–50
d
15–50
d
a
40 laboratories reporting
b
39 laboratories reporting
c
7 laboratories reporting
d
Some laboratories reported a minimum read depth of 15 and will visually inspect reads of 15–50 before determining whether to perform Sanger sequencing
analysis. Minimum refers to the minimum at which no additional studies are performed
Fig. 4 Turn-around time per testing volume. The turn-around time
(TAT) in days is plotted as a function of number of BRCA1/BRCA2 tests
performed in a one-year time period for the three US laboratories
and 38 non-US laboratories who reported values for both questions.
US laboratories are indicated by a circle. No correlation was found
between TAT and testing volume for all 41 laboratories or the 38
non-US laboratories (Pearson’s correlation =−0.23 and 0.02
respectively)
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AE Toland et al.
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npj Genomic Medicine (2018) 7 Published in partnership with the Center of Excellence in Genomic Medicine Research

numbers of patients referred for BRCA1/BRCA2 testing. There was a
strong correlation for US laboratories between TAT and number of
BRCA1/BRCA2 tests performed annually (Pearson’s correlation =
−0.76); however there was no correlation for non-US laboratories
(Pearson’s correlation = 0.02).
Although we aimed to do a global survey, a limited number of
laboratories from Africa, South America, and Asia were sent direct
survey links. As the survey was in English, language differences
also may have contributed to a low response rate for laboratories
in Asian, African, and South American countries. Additionally, as
the survey was lengthy and some questions required additional
research, some laboratories may not have participated. The US-
response rate was low (8 of 27), but because some of the surveys
to the non-US laboratories were sent by others through laboratory
networks, we are unable to calculate response rate. This survey
was not comprehensive and may be missing information on some
key differences in practices, such as method of library preparation
for MPS and rationale for choice of technologies utilized.
Additionally, this survey was not designed to compare sensitivity
and specificity of pathogenic variant detection or VUS calling
which is an important consideration for establishing best
practices. Finally, we did not collect information on the rationale
behind the choice of technologies for each laboratory which could
be useful for laboratories updating technologies for BRCA1/2
variant identification.
In conclusion, this study shows that there are key similarities in
technology used for BRCA1/BRCA2 sequence and rearrangement
analysis around the world. There was variation in laboratory-
specific quality criteria for average and minimum numbers of read
depths which could impact sensitivity. Laboratories reported VUS
at varying rates, but also calculated rates of VUS differently. Based
on these results and two recent studies, one showing a 2.6%
diagnostic error rate for detection of pathogenic and significant
variants using MPS approaches,
11
and a second study suggesting
that only two of seven MPS workflows could detect all
23 “challenging variants”in a blinded study
12
, we suggest points
for consideration for laboratories performing or contemplating
BRCA or panel testing (Fig. 5). These points for consideration (Fig.
5) are complemented by those from other groups, such as the
Association for American Pathologist and the College of American
Pathologist who have developed standards and guidelines for
MPS Bioinformatics pipelines used in clinical tests.
13
Of critical
note, global data sharing of variants with sufficient data to allow
multiple lines of evidence without duplication would facilitate
variant reclassification and a central alert system to improve the
quality of variant analysis and ensure more consistent manage-
ment of high risk gene carriers as has been highlighted
recently.
14,15
Due to the diversity across laboratories, this study
highlights the opportunity for international organizations to
formulate guidelines for laboratories performing BRCA1/BRCA2
genetic testing.
MATERIALS AND METHODS
Survey
Two online surveys were developed: one for non-US laboratories and one
for US laboratories that included questions specific to the US (Supple-
mentary Notes 1 and 2). Each survey had four sections: 1: Testing
Laboratory Information, 2: Multigene Hereditary Cancer Panels, 3: Variant
Assessment; and 4: Staffing, Billing and Other Client-Support Related
Questions. Surveys were developed using Qualtrics Survey Software
(Qualtrics, Provo, UT and Seattle, WA).
Distribution of survey
Initial links to surveys were sent on 1 December 2016. Contact names and
potential testing laboratories were identified through the BIC Steering
Committee contacts, Google searches and mass e-mails sent from
coordinators of the European Molecular Genetics Quality Network and
United Kingdom National External Quality Assessment Service for
Molecular Genetics to all genetics testing laboratories in their networks.
Twenty-seven US laboratories were sent the survey. Survey links were
distributed over a 4-month period. The international survey closed to new
respondents on 29 March 2017 and the US survey closed to new
respondents on 9 May 2017. For the non-US laboratories, 78 laboratories
answered some questions, and 48 of those fully completed the survey,
although some questions were not applicable to all laboratories
(Supplementary Table 1). Eight US laboratories responded to the survey
(Supplementary Table 2). The number of laboratories answering each
question is included for each data point as the denominator varies.
Data analysis
Summary data and frequencies were tallied in Qualtrics. As some questions
had “click all that apply”answers, the percentage and number of
laboratories responding are both reported. When a range of values was
reported (e.g., 500–1000 read depth), the mid-point was used in the
calculation (e.g., 750 reads). When a greater than or less than symbol was
included (e.g., >100), the numerical value was used in the calculation.
Answers related to time were converted to weeks.
Map generation
The geographical locations of the participating laboratories were mapped
by plotting the latitude and longitude markers obtained from the IP
address of the individual completing the survey using ZeeMaps (https://
www.zeemaps.com).
Data availability
The authors declare that all of the data supporting the findings of this
study are available within the paper and its supplementary information
files.
ACKNOWLEDGEMENTS
We thank the laboratory personnel and directors for taking the time to complete
these surveys. Simon Patton and Zandra Deans kindly distributed the survey link for
this study to their clinical laboratory networks: EMQN and UK NEQAS for Molecular
Genetics. David Kaufman, NHGRI provided advice on the structure of the survey. The
BIC steering committee provided thoughtful discussion on survey questions and data
presentation and international laboratory contacts. This work was supported in part
by the Intramural Research Program of the National Human Genome Research
Institute and by the Intramural Research Program, Center for Cancer Research,
National Cancer Institute. A.N.M. is funded by the Florida Breast Cancer Foundation. S.
L.N. is the Morris and Horowitz Families Endowed Professor. A.B.S. is funded by a
National Health and Medical Research (NHMRC) Senior Research Fellowship. W.D.F. is
funded by the Canadian Institute for Health Research.
AUTHOR CONTRIBUTIONS
All authors designed the study and the survey. Surveys were distributed by A.F. and
A.E.T. A.E.T. analyzed and curated the data. A.E.T. and A.F. wrote the manuscript: All
authors edited the manuscript and approved the final paper. All authors have
accountability for all aspects of work.
Fig. 5 Points to consider. Points to consider which may improve
client satisfaction and/or facilitate characterization of VUS are listed
Clinical testing of BRCA1 and BRCA2
AE Toland et al.
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Published in partnership with the Center of Excellence in Genomic Medicine Research npj Genomic Medicine (2018) 7

ADDITIONAL INFORMATION
Supplementary information accompanies the paper on the npj Genomic Medicine
website (https://doi.org/10.1038/s41525-018-0046-7).
Competing interests: A.F. is a paid advisor and speaker for Ambry and Invitae. S.E.P.
is on the scientific advisory board of Baylor Genetics. The remaining authors declare
no competing financial interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
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npj Genomic Medicine (2018) 7 Published in partnership with the Center of Excellence in Genomic Medicine Research