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A multi-center prospective cohort study of the diagnostic yield and patient experience of multiplex gene panel testing for hereditary cancer risk

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A multi-center prospective cohort study of the diagnostic yield and patient experience of multiplex gene panel testing for hereditary cancer risk

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Content 1

Title: A Multi-Center Prospective Cohort Study of the
Diagnostic Yield and Patient Experience of Multiplex
Gene Panel Testing For Hereditary Cancer Risk

Candidate: Gregory Idos MD

Thesis Advisor: Stephen Gruber MD, PhD, MPH

Conferring Major/Program: Clinical, Biomedical, and
Translational Research/Preventive Medicine

Degree being conferred: Master of Science

UNIVERSITY OF SOUTHERN CALIFORNIA

Degree conferral date (anticipated): December 12,
2018

2

Table of Contents: PAGE
Title Page------------------------------------------------------------------------------------------------------------1
Table of Contents-------------------------------------------------------------------------------------------------2
Body Text
Introduction-----------------------------------------------------------------------------------------------3
Methods---------------------------------------------------------------------------------------------------4-8
Summary Results------------------------------------------------------------------------------------8-16
Discussion---------------------------------------------------------------------------------------------16-21

APPENDICES:
App1: Genes analyzed by the multiple gene hereditary cancer panel ----------------22
App2: CONSORT diagram of patient enrollment and outcomes------------------------23

App3: Prophylactic Surgery among patients with high-penetrance mutations
and low-penetrance mutations---------------------------------------------------------------------------24-25

References------------------------------------------------------------------------------------------------------26-28

3

INTRODUCTION:
Genetic testing is a powerful tool to stratify cancer risk. Recent advances in
massively parallel sequencing have expanded germline testing for hereditary cancer
risk assessment.(1-3) Multiplex gene panel testing (MGPT) simultaneously analyzes
multiple genes, and may provide an advantage over sequential single-gene testing in
terms of cost, speed and clinical utility. Due to the wide spectrum and considerable
overlap of phenotypes associated with hereditary cancer syndromes, MGPT offers a
potentially practical and efficient approach to identifying the genetic cause of inherited
cancer susceptibility.(4-13)
Despite these advantages, MGPT has raised concerns about the identification of
pathogenic variants that do not correlate with phenotype (for example, BRCA1 or
BRCA2 mutations in patients clinically suspected to have Lynch Syndrome).(14)
Furthermore, MGPT yields 10-fold higher rates of clinically ambiguous variants of
uncertain significance (VUS) compared to more limited testing approaches.
(2-13, 15, 16)

Very little is known about the effect of this greater volume of uncertain results on the
outcomes and patient experience of cancer risk assessment, and it is crucial to learn
more as more comprehensive sequencing approaches on the horizon.
We designed a prospective cohort study of hereditary cancer testing with a multi-
gene panel to measure the benefits, harms, and patient experiences of MGPT. Our
hypotheses were that MGPT with pre-test genetic counseling would be associated with:
1) patient regret, 2) utilization of preventive surgery, and 3) family communication of
results. We report the diagnostic yield and patient experience of MGPT after full accrual
of the planned 2000 participants.

4

METHODS:
Study Population
The study was approved by institutional review boards (protocol #HS-13-00431)
at the University of Southern California (USC) and Stanford University. Written informed
consent was obtained in person from each subject. Participants were consecutively
recruited between July 2014 and November 2016 at three medical centers: 1) USC
Norris Comprehensive Cancer Center; 2) Los Angeles County + USC Medical Center;
3) Stanford University Cancer Institute. Eligible patients met clinical guideline criteria for
genetic testing or had a ≥2.5% probability of mutation carriage calculated by the
following validated models or algorithms: BOADICEA, BRCAPro, IBIS (Tyrer-Cuzick),
PANCPro, PREMM1,2,6, PENN II, PTEN Cleveland Clinic Score, MELAPro, MMRPro,
Myriad II, National Comprehensive Cancer Network (NCCN) Guidelines, or a personal
history of >10 cumulative lifetime colorectal adenomas.

Next Generation Sequencing (NGS) Assay
MGPT was accomplished with a multiple-gene, next-generation sequencing test
performed by Myriad Genetic Laboratories (Salt Lake City, UT), including APC, ATM,
BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM,
GREM1, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, POLD1, POLE, PTEN,
RAD51C, RAD51D, SMAD4, STK11, and TP53 (Appendix 1 in Supplement). All genes
were included over the full study period except for GREM1, POLD1, and POLE, which
were added in July 2016. There were 1664 participants who underwent 25-gene panel
testing and 336 who underwent 28-gene panel testing. Sequencing and large
rearrangement analysis was performed for all genes except for the following: POLD1
5

and POLE (sequencing only), EPCAM and GREM1 (large rearrangement only).
Sample preparation for NGS was performed from frozen DNA using the
RainDance microdroplet polymerase chain reaction (PCR) system (RainDance
Technologies, Billerica, MA).
(17)
Briefly, PCR products representing exons and proximal
splicing elements of patient DNA were amplified in merged droplets consisting of
fragmented patient DNA and select target enrichment primers. These PCR products
were subsequently tagged with barcodes and sequencing adaptors for NGS on the
Illumina HiSeq platform (Illumina, San Diego, CA). To circumvent highly homologous
pseudogenes, modified sample preparation with long-range and nested PCR, followed
by NGS on the Illumina MiSeq platform, was used for portions of CHEK2 and PMS2. All
clinically actionable variants identified by NGS, as well as regions that did not meet
preset NGS quality metrics, were independently confirmed with orthogonal site-specific
Sanger sequencing. To detect exonic deletions and duplications, NGS dosage,
microarray comparative genomic hybridization, multiplex ligation-dependent probe
amplification, or a combination of these analyses was performed, with all positive results
confirmed by an orthogonal method.
(17)

Variant Classification
Variants were classified using American College of Medical Genetics and
Genomics recommendations, with supporting linkage, biochemical, clinical, functional,
and statistical data used for specific missense and intronic alterations.(18-20) Gene
variants classified as deleterious or suspected deleterious were considered pathogenic.
Variants with unknown clinical significance were considered variants of uncertain
6

significance (VUS). Variants classified as polymorphism or favor polymorphism were
considered benign.

Differential Diagnoses
Differential diagnoses were generated for each participant after expert clinical
genetics assessment, in which up to eight inherited cancer syndromes were ranked by
estimated likelihood using factors such as personal and family cancer history, tumor
characteristics, and physical examination (eTable 1).
(21)
The genetics clinician then
clarified a level of suspicion for each syndrome in the differential diagnosis by stating
whether s/he would test for that syndrome specifically if MGPT were not available.

Questionnaire Procedures
Participants were invited to answer a baseline questionnaire at the time of their
genetics evaluation, with follow-up questionnaires three, six and twelve months after
MGPT result disclosure. Participants were contacted by mail and/or electronic mail
(email) to complete follow-up questionnaires, which included a brief reminder of the
study’s purpose and procedures. All mailed questionnaires included a postage-paid,
pre-addressed envelope. Email participants who did not respond to the initial invitation
link received two email reminders over a course of two weeks before receiving a
reminder phone call from study personnel. Mail participants were given two weeks to
respond, after which they received a reminder phone call. Using a standardized script,
all participants who had a reminder phone call were given the option to receive another
email invitation, mailed paper questionnaire, or to complete the questionnaire over the
7

phone. A maximum of three phone attempts for contacting the participant, including
leaving voicemails, were made by study personnel. Participants were considered non-
responders if they had not completed their questionnaire two months after the initial
send date.

Patient-Reported Experiences
We used the validated Multidimensional Impact of Cancer Risk Assessment
(MICRA)(22) instrument, which contains subscales that measure distress, uncertainty,
and positive experiences in relation to genetic testing. The distress subscale (6 items,
score range 0-30) evaluates adverse psychological feelings of anxiety and regret. The
uncertainty subscale (9 items, score range 0-45) evaluates doubt and frustration. The
positive experience subscale (4 items, score range 0-20) evaluates relief and
satisfaction. Additional questions evaluated intrusive thoughts about cancer and regret
about having undergone MGPT, participants’ desired amount of MGPT results
information, participants’ notification of relatives about MGPT results and relatives’
genetic testing behaviors. Participants were also asked if they had undergone specific
surgical procedures (mastectomy, salpingo-oophorectomy, hysterectomy) and the
reason for these procedures (cancer treatment, cancer prevention, other).(23)

Statistical Analysis
The primary aim of this study was to test the impact of genetic test results
(positive, VUS, negative) on patient experience. The MICRA subscale scores were used
for this aim. Patients with ≥1 pathogenic variant were considerd ‘positive’ while those
8

with only benign variants were considerd ‘VUS’ and those with no variants were
considered ‘negative’. Sample size estimation was based on comparisons between
genetic test results. Assuming a pathogenic variant prevalence of 10%, a VUS
prevalence of 35%, and a negative test prevalence of 55%, and the standard deviation
of MICRA scores being 4, a sample size of 2000 patients was needed to achieve >80%
power to detect a difference of 1 in MICRA scores.
Data analysis was based on information gathered as of March 19, 2018.
Descriptive statistics were calculated for demographics, differential diagnoses, surgery
and MGPT results. The Pearson chi-square test was used to assess the association
between genetic test results and survey responses. Negative binomial regression with a
log link was used to analyze the association of genetic test results with the MICRA
subscale scores while adjusting for covariates including clinical site, age, gender,
ethnicity, education level, and personal history of cancer. P-values less than 0.05 were
considered statistically significant. All statistical analyses were performed with SAS
software, version 9.4 (Cary, N.C.) and R software version 3.2.2.

SUMMARY RESULTS:
Study Population
Two thousand participants were enrolled between July 2014 and November 2016
(eFigure 1 in Supplement). The majority (81%) were female and 73% had a personal
history of cancer (Table 1). Common reasons for genetics referral include the following:
cancer diagnosis under the age of 50 (39%), ≥2 first or second-degree relatives with
cancer (64%), ≥1 family member who was diagnosed with cancer under age 50 (63%).
9

Table 1. Participant Characteristics by Clinical Site

Characteristic
USC Norris
(N=797)
LAC+USC
(N=715)
Stanford
(N=488)
p-
Value*
Total
(N=2000)
Age at testing (years)
Mean 50.7 49.5 55.6 <0.001 51.5
SD 14.91 10.18 13.95 13.37
Median 51 49 57 51
Min, Max 16, 92 21, 92 17, 90 16, 92
Gender <0.001
Female 591 (74.2%) 610 (85.3%) 413 (84.6%) 1614 (80.7%)
Male 206 (25.8%) 105 (14.7%) 75 (15.4%) 386 (19.3%)
Primary language <0.001
English 702 (88.2%) 183 (25.7%) 419 (85.9%) 1304 (65.4%)
Spanish 23 (2.9%) 462 (65.0%) 34 (7.0%) 519 (26.0%)
Other 71 (8.9%) 66 (9.3%) 35 (7.2%) 172 (8.6%)
Race/Ethnicity <0.001
Non-Hispanic, White 460 (57.7%) 53 (7.4%) 294 (60.2%) 807 (40.4%)
Hispanic, White 151 (18.9%) 547 (76.5%) 83 (17.0%) 781 (39.1%)
Asian 104 (13.0%) 65 (9.1%) 65 (13.3%) 234 (11.7%)
Black or African American 34 (4.3%) 32 (4.5%) 10 (2.0%) 76 (3.8%)
Other† 48 (6.0%) 18 (2.5%) 36 (7.4%) 102 (5.1%)
Education <0.001
High school or less 83 (10.4%) 444 (62.1%) 74 (15.2%) 601 (30.1%)
Some college 144 (18.1%) 111 (15.5%) 106 (21.7%) 361 (18.1%)
College degree or more 419 (52.6%) 90 (12.6%) 288 (59.0%) 797 (39.9%)
Other or missing‡ 151 (18.9%) 70 (9.8%) 20 (4.1%) 241 (12.1%)
Personal cancer history§ <0.001
Affected 549 (68.9%) 554 (77.5%) 348 (71.3%) 1451 (72.6%)
Unaffected 248 (31.1%) 161 (22.5%) 140 (28.7%) 549 (27.4%)
Reasons for genetic evaluation‖
Diagnosed with cancer <50
years old
280 (35.1%) 364 (50.9%) 143 (29.3%) <0.001 787 (39.4%)
≥2 first or second degree
relatives with cancer
518 (65.0%) 431 (60.3%) 335 (68.6%) 0.010 1284 (64.2%)
≥1 family member
diagnosed with cancer
<50 years old
404 (50.7%) 583 (81.5%) 276 (56.6%) <0.001 1263 (63.2%)
History of multiple primary
cancers
102 (12.8%) 68 (9.5%) 99 (20.3%) <0.001 269 (13.5%)
Abbreviations: USC Norris, University of Southern California Norris Comprehensive Cancer Center;
LAC+USC, Los Angeles County and University of Southern California Medical Center; SD, standard
deviation; Min, minimum value; Max, maximum value.
*p-Values for comparison between clinical sites using analysis of variance for age at testing and the chi-
square test for other characteristics.
†Other Race category includes American Indian/Alaska Native, Native Hawaiian or Pacific Islander, More
than one race, and Unknown.
‡Other Education category includes trade/vocational school.
§Excluding non-melanoma skin cancer.
‖Subjects could note more than one reason.
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Participants were diverse both racially/ethnically and sociodemographically: 39%
were Hispanic (primarily of Mexican or Central American ancestry), 40% non-Hispanic
White,12% Asian (primarily of Chinese and Filipino ancestry), and 4% Black.
Approximately one-quarter (26%) spoke Spanish as their primary language and 30%
had completed high school or less education (Table 1).

Frequency of Pathogenic Variants and Variants of Uncertain Significance
At least one pathogenic variant was identified in 242 (12%) participants (Figure
1). Seventy-six patients (31% of all pathogenic variant carriers) had a germline mutation
in BRCA1 and/or BRCA2, and 39 (16%) had a pathogenic variant in a mismatch repair
gene conferring a diagnosis of Lynch Syndrome. Forty-three (18%) had a pathogenic
MUTYH variant: monoallelic (n=41), biallelic (n=2). Nineteen patients (8%) had
pathogenic variants in APC, with sixteen of them having the founder mutation APC
I1307K. Six patients had pathogenic variants in TP53 (2%). Other genes in which
pathogenic variants were detected included CHEK2 (n=17, 7%), ATM (n=16, 7%),
PALB2 (n=9, 4%), BRIP1 (n=5, 2%), RAD51C (n=4, 2%), BARD1 (n=2, 1%), NBN (N=2,
1%), CDH1 (n=1, 0.4%) and CDKN2A (n=1, 0.4%). Pathogenic variant status and
associated patient characteristics are shown in Table 2. Among patients without
pathogenic variants, 689 (34%) had at least one VUS, with up to four per patient (Table
2 ).

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Figure 1. Overall yield of genetic testing among 2000 participants

12

Table 2. Sociodemographic and Clinical Characteristics by Genetic Test Result
Characteristic
Positive
(N=242)
Negative
(N=1069)
VUS
(N=689)
Total
(N=2000)
Clinical site
USC Norris 91 (37.6%) 430 (40.2%) 276 (40.1%) 797 (39.9%)
LAC+USC 94 (38.8%) 385 (36.0%) 236 (34.3%) 715 (35.8%)
Stanford 57 (23.6%) 254 (23.8%) 177 (25.7%) 488 (24.4%)
Age at testing (years)
Mean 52.3 51.1 51.7 51.5
SD 12.89 13.37 13.52 13.37
Median 53 51 51 51
Min, Max 22, 89 16, 92 16, 92 16, 92
Gender
Female 189 (78.1%) 867 (81.1%) 558 (81.0%) 1614 (80.7%)
Male 53 (21.9%) 202 (18.9%) 131 (19.0%) 386 (19.3%)
Race/Ethnicity
Non-Hispanic, White 101 (41.7%) 463 (43.3%) 243 (35.3%) 807 (40.4%)
Hispanic, White 97 (40.1%) 423 (39.6%) 261 (37.9%) 781 (39.1%)
Asian 27 (11.2%) 82 (7.7%) 125 (18.1%) 234 (11.7%)
Black or African American 10 (4.1%) 34 (3.2%) 32 (4.6%) 76 (3.8%)
Other* 7 (2.9%) 67 (6.3%) 28 (4.1%) 102 (5.1%)
Education
High school or less 79 (32.6%) 325 (30.4%) 197 (28.6%) 601 (30.1%)
Some college 45 (18.6%) 196 (18.3%) 120 (17.4%) 361 (18.1%)
College degree or more 83 (34.3%) 423 (39.6%) 291 (42.2%) 797 (39.9%)
Other or missing† 35 (14.5%) 125 (11.7%) 81 (11.8%) 241 (12.1%)
Personal cancer history‡
Affected 190 (78.5%) 755 (70.6%) 506 (73.4%) 1451 (72.6%)
Unaffected 52 (21.5%) 314 (29.4%) 183 (26.6%) 549 (27.5%)
Abbreviations: VUS, variant of uncertain significance; USC Norris, University of Southern California Norris
Comprehensive Cancer Center; LAC+USC, Los Angeles County and University of Southern California
Medical Center; SD, standard deviation; Min, minimum value; Max, maximum value.
*Other Race category includes American Indian/Alaska Native, Native Hawaiian or Pacific Islander, More
than one race, and Unknown.
†Other Education category includes trade/vocational school.
‡Excluding non-melanoma skin cancer.

Differential Diagnosis versus MGPT Results
Of 242 participants with pathogenic variants, 160 (66%) had pathogenic variants
in genes related to syndromes included in the pre-test differential diagnosis. Eighty-two
patients (34%) had pathogenic variants that were not clinically anticipated: monoallelic
MUTYH (n=32, 39%), APC I1307K (n=13, 16%), CHEK2 (n=10, 12%), PALB2 (n=8,
10%), ATM (n=7, 9%), BRIP1 (n=4, 5%), BRCA2 (n=3, 4%), BRCA1 (n=2, 2%), PMS2
(n=2, 2%) and TP53 (n=1, 1%).
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Use of Surgery after MGPT
At a median follow up of 13 months, 198 of 1488 (13%) returning surveys
reported undergoing surgery after MGPT. Surgery rates and stated reasons were as
follows: mastectomy (n=162, 11.3%: 93% cancer treatment, 30% cancer prevention, 2%
benign breast disease), salpingo-oophorectomy (n=43, 3%: 27% cancer treatment, 56%
cancer prevention), hysterectomy (n=23, 2%: 50% cancer treatment, 18% cancer
prevention, 9% benign disease). Overall, only 4% (n=62) of patients underwent a
prophylactic operation. Significantly more patients who tested positive (n=30, 16%) had
preventive surgery compared either to patients testing negative (n=20, 2.4%, p<0.001)
or with a VUS (n=12, 2.3%, p<0.001). There was no significant difference in use of
preventive surgery between those testing negative as compared to VUS (p=0.919). To
illustrate, there were ten patients identified as having high-penetrance founder
mutations in BRCA1 or BRCA2 , six of whom were found to have undergone or were
considering risk reducing mastectomy or oophorectomy. In comparison, there were 16
patients identified as carriers of the low penetrance APCI1307K allele, none of whom
had undergone an inappropriate surgery. (eTable 2 in Supplement)

Patient Experiences with MGPT
At a median follow-up of four months, the response rate was 69%. Overall, levels
of genetic testing-specific concerns were low. Mean scores for each MICRA sub-scale
(distress, uncertainty, positive experiences) all differed significantly between patients
with positive test results compared to the group of patients with a negative or VUS result
14

(Table 3). When compared to patients testing negative or VUS, patients testing positive
had significantly higher distress scores (p<0.001 or p<0.001, respectively), significantly
higher uncertainty scores (p<0.001 or p<0.001, respectively) and significantly lower
positive experiences scores (p<0.001 or p=0.007, respectively). When compared to
patients testing negative, patients with a VUS had significantly higher uncertainty scores
(p=0.017) but not significantly different distress or positive experiences scores (p=0.249
or p=0.399, respectively).
Two-thirds (67%) of patients stated that thoughts of cancer rarely or never
affected their daily activities, most (92%) never regretted learning their MGPT results,
and most (80%) wanted to know all MGPT results, including findings that doctors do not
fully understand (Figure 2). Responses to these questions did differ significantly
between positive and negative patients, with the exception of wanting to know all MGPT
results. Positive patients were less likely to have thoughts of cancer affect their daily
activites (55% vs. 70%, p<0.001) or regret learning their test results (85% vs. 95%,
p<0.001) (Figure 2.). However, all were similarly likely (96–99%) to notify relatives about
results. There were significant differences (p<0.001) in family communication and
encouragement of relatives’ testing between positives (for whom relatives’ testing is
strongly indicated)
21-22
versus the VUS or negative group (for whom relatives’ testing is
sometimes indicated)
21-22
as follows: encouraged relatives to have genetic testing
(positive 83%, VUS 43%, negative 40%), relatives underwent genetic testing (positive
38%, VUS 6%, negative 5%), and relatives underwent screening because of increased
cancer risk (positive 45%, VUS 23%, negative 19%).

15

Figure 2. Patient experiences after multiplex testing

16

Table 3. Multidimensional Impact for Cancer Risk Assessment (MICRA) Subscale
Score Results by Genetic Test Result
MICRA Subscale
Positive
(N=242)
Negative
(N=1069)
VUS
(N=689)
p-Value*
Total
(N=2000)
Distress (Possible Scores Range from 0-30)
n 163 669 416 1248
Mean 6.1 1.7 2.1 2.4
SD 6.04 3.54 4.22 4.41
Minimum 0 0 0 PN<0.001 0
Q1 1 0 0 PV<0.001 0
Median 4 0 0 VN=0.249 0
Q3 10 2 3 3
Maximum 26 28 30 30
Uncertainty (Possible Scores Range from 0-45)
n 161 655 407 1223
Mean 11.4 6.3 7.4 7.3
SD 8.75 7.13 7.78 7.75
Minimum 0 0 0 PN<0.001 0
Q1 5 1 1 PV<0.001 1
Median 10 4 5 VN=0.017 5
Q3 17 9 11 11
Maximum 41 35 43 43
Positive Experiences (Possible Scores Range from 0-20)
n 158 650 405 1213
Mean 9.7 12.1 11.8 11.7
SD 5.06 6.46 6.33 6.29
Minimum 0 0 0 PN<0.001 0
Q1 6 8 8 PV=0.007 8
Median 10 12 12 VN=0.399 12
Q3 12 18 16 16
Maximum 20 20 20 20
Abbreviations: VUS, variant of uncertain significance; SD, standard deviation; Q1, first quartile; Q3, third
quartile; PN, Positive vs Negative; PV, Positive vs VUS; VN, VUS vs Negative
*p-Values for comparison between genetic test results using negative binomial regression with a log link
and adjusting for clinical site, age at testing, gender, race, personal cancer history and education.

DISCUSSION:
This prospective cohort study of MGPT for hereditary cancer risk presents data
that inform current practice of genetic testing. The results offer a window into patient
perceptions of a future driven by increasingly complex and rich genetic information.
Among patients who carried a pathogenic variant (12%), one-third of these clinically
relevant results were not suspected upon pre-test expert assessment. We found little
evidence of patient harm and substantial evidence that patients understood the
17

meaning of their MGPT results, as patients who tested positive for a pathogenic variant
were more likely to encourage appropriate genetic testing in relatives (compared to
those who tested VUS/negative). These results suggest an important contribution of
MGPT to clinical cancer risk assessment of probands and their families.
A striking result was that one in three identified pathogenic variants was missed
in the differential diagnosis generated by expert clinicians. This finding highlights the
limitations of clinical risk assessment and the incremental diagnostic yield from testing
genes beyond those implicated in a single syndrome. Notably, few of the missed
variants were in genes associated with well-known syndromes (e.g., Lynch Syndrome,
Hereditary Breast/Ovarian Cancer), which likely reflects clinicians’ greater familiarity
with their presentation. Instead, most were in “moderate-penetrance” genes that confer
a two- to four-fold increase in cancer risk (e.g., ATM and CHEK2), and “low-penetrance”
mutations (e.g., monoallelic MUTYH and APC I1307K). Given the short history of
widespread clinical testing of these genes,(24-27) their phenotype is less well
characterized and more difficult for clinicians to recognize. Moreover, our relatively low
threshold for study eligibility (estimated risk of pathogenic variant probability ≥2.5%,
approximately half that of practice guidelines(28, 29), chosen using published cost-
effectiveness estimates at different risk thresholds(30)) may have enriched for less
penetrant variants. Some questions remain about the clinical benefit of detecting low- to
moderate-penetrance pathogenic variants; however, these variants meet criteria for
intensified cancer screening according to current practice guidelines,(28, 29) which
makes them relevant to patient care. By identifying unexpected pathogenic variants,
MGPT can broaden our understanding of genotype-phenotype correlations and the
18

spectrum of associated cancer risks.
Concerns have been raised about high rates of identifying VUS in MGPT.
Consistent with prior studies, we report a VUS rate of 34%.
5-8
Recent studies found
limited “genomic confidence” among oncologists, and that few (<15%) community
physicians who order BRCA1 and BRCA2 tests understand the correct management of
VUS.(27, 31, 32) We recently published that patients who receive VUS results from
genetic testing are substantially more likely to seek a second opinion from a new
medical oncologist.(33) A related concern is that MGPT results of unknown clinical
relevance, whether VUS or a pathogenic variant whose cancer risk is insufficiently
characterized, might prompt invasive and irreversible prophylactic operations.(34-36)
Data from our prospective cohort study do not support this hypothetical concern.
Reassuringly, we found that prophylactic operations were not overutilized: only 4% had
prophylactic mastectomy, hysterectomy, or salpingo-oophorectomy, and these
procedures were no more frequent among patients with VUS than negative results. With
a median follow-up time of 13 months after genetic testing, these results do not suggest
an over-utilization of surgery after MGPT in this study.
Notably, the overall yield of pathogenic variants in this study is 12%, which is
similar to other studies. While panels may vary in the number of genes tested, all large
panels, including the one used in this study, include genes associated with known
syndromes (i.e. HBOC and Lynch syndrome), which account for the majority of
pathogenic mutations identified in most multigene panel studies. Our group previously
examined the additional yield of mutations identified via multigene panel testing and
found that by increasing the number of genes tested, the frequency of mutations
19

identified also increases.(37, 38) Independently, a recent study by Mandelker et al.(39)
demonstrated a 17.5% yield of pathogenic variant yield after sequencing 1040 patients
with advanced cancer using the MSK-Impact panel of 410 genes.
Patient-reported experiences were also reassuring. Most never regretted MGPT
or had intrusive thoughts about cancer, and most wanted to know all MGPT results,
even those that physicians do not fully understand. This is concordant with prior studies
showing that patients consistently express a desire to know genetic test results in a
multitude of settings.(40) We found that MICRA subscale scores varied significantly
between patients who tested positive compared to those who had a negative or VUS
test result, but scores did not vary between patients with a VUS versus negative except
in the category of uncertainty. Patients also communicated differently to relatives: those
who tested positive were twice as likely to urge their relatives to be tested, which is
guideline-concordant and appropriate management for a positive result. These findings
suggest that patients rarely misinterpret VUS with appropriate pre-test genetic
counseling since most VUS that are reclassified are ultimately reclassified to
benign.(15, 41) On the contrary, these findings suggest that patients understood the
implications of their MGPT results, which is particularly encouraging in our population
since approximately one-third had a high school education or less. The present results
contrast with our recent finding of gaps in the understanding and management of VUS
in community practice, and serve to demonstrate the value of pre- and post-test
counseling with proper anticipatory guidance by a trained professional.(42-44)
There are limited published data on pathogenic mutation rates among Hispanics
after multiplex gene panel testing, as Hispanics are underrepresented in a majority of
20

clinical studies. Notably, we found that 12.1% (n=97/781) of Hispanic patients had a
pathogenic variant. In our previous USC-based paper by Ricker et al.(37) where
Hispanic patients made up 47.6% (n=228) of the cohort, there was a pathogenic
mutation frequency of 14.1% (n=33). In a recent publication from Stanford, Caswell-Jin
et al.(45) reported that 21% of their 213 Hispanic participants carried a pathogenic
variant. In a study by Rosenthal et al.(11), among the 8% (n=19,795) of Hispanic
patients that underwent multigene panel testing, 6.7% (n=1326) were found to have a
pathogenic mutation. More data are needed to understand the rates of pathogenic
mutations in different ethnic groups.
Aspects of our study merit comment. Its considerable strengths include a
prospective, multi-center design; a diverse population in terms of race/ethnicity,
language and education; a high survey response rate; and uniform pre-test assessment
by experienced genetic counselors. The participation rate of Hispanic/Latino, African
American, and Asian patients was very high compared to other published studies. It is
the first study to demonstrate that testing at lower predicted levels of pathogenic variant
carriage, which reflects recent changes in clinical practice and guidelines, is effective
and safe. This study offers significant and novel results about the performance of
multiplex genetic testing and has broad implications for its clinical implementation and
acceptance. Looking forward, interim analysis of our data suggests that patients found
to have a pathogenic variant in a high or moderate risk gene are more likely to undergo
appropriate screening and surveillance as compared to those who tested negative or
with a VUS within 1 year of MGPT testing. For example, patients identified to carry a
pathogenic variant in a gene associated with Lynch syndrome were 4 times as likely
21

(p<0.001) to undergo colonoscopy as compared to patients with a VUS or negative
genetic test result. Encouragingly, this trend is consistent when we stratify by ethnicity in
our Hispanic cohort (manuscript in preparation).
Its limitations include 13 months of follow-up time to date. In addition, our survey
response rate decreased to 57% at 12 months from 72% at 3 months as we primarily
recruited from a clinical cohort, most of whom were affected with cancer and undergoing
active treatment. During the course of follow-up, some patients passed away, moved to
another location, or changed their contact information. Nonetheless, this fully accrued,
prospective study of 2000 participants has immediate relevance for patient care.
In conclusion, the results of this prospective cohort study support the use of
multiplex gene panel testing for hereditary cancer risk assessment with appropriate
genetic counseling, and demonstrate its capacity to enhance the diagnostic yield of
genetic testing without discernible harm to patients. Longer-term follow-up of clinical
and patient-reported outcomes will further inform the implementation of increasingly
comprehensive genetic testing into clinical practice.

22

APPENDICES:
Appendix 1. Genes analyzed by the multiple gene hereditary cancer panel
Gene Syndrome Major Cancers
APC Familial Adenomatous Polyposis Syndrome Colon
ATM Ataxia Telangiectasia Breast
BARD1 Hereditary Breast and Ovarian Cancer Breast, ovarian
BMPR1A Juvenile Polyposis Syndrome Gastrointestinal
BRCA1 Hereditary Breast and Ovarian Cancer Breast, ovarian
BRCA2 Hereditary Breast and Ovarian Cancer Breast, ovarian
BRIP1 Hereditary Breast and Ovarian Cancer Ovarian, possibly breast
CDH1 Hereditary Diffuse Gastric Cancer Breast, gastric
CDK4 Hereditary Melanoma (Multiple Nevi Syndrome) Melanoma
CDKN2A
a
Hereditary Melanoma (Multiple Nevi Syndrome) Melanoma, pancreatic
CHEK2 Breast and Colon Cancer Breast, colon
EPCAM Lynch Syndrome Colon, endometrial, ovarian
GREM1
b
Polyposis Colon
MLH1 Lynch Syndrome Colon, endometrial, ovarian
MSH2 Lynch Syndrome Colon, endometrial, ovarian
MSH6 Lynch Syndrome Colon, endometrial, ovarian
MUTYH MYH-associated polyposis Colon
NBN Hereditary Breast and Ovarian Cancer Breast, possibly ovarian
PALB2 Pancreatic and Breast Cancer Breast, pancreatic
PMS2 Lynch Syndrome Colon, endometrial, ovarian
POLE
b
Polyposis Colon
POLD1
b
Polyposis Colon
PTEN Cowden Syndrome
Breast, endometrial, renal,
thyroid
RAD51C Hereditary Breast and Ovarian Cancer Breast, ovarian
RAD51D Hereditary Breast and Ovarian Cancer Breast, ovarian
SMAD4 Juvenile Polyposis Syndrome Gastrointestinal
STK11 Peutz Jeghers Syndrome Breast, gastrointestinal
TP53 Li-Fraumeni
Adrenal, brain, breast, colon,
leukemia, sarcoma, others
a
p16INK4a and p14ARF
b
Added to multi-gene panel in July 2016

23

Appendix 2: CONSORT diagram of patient enrollment and outcomes
24
Appendix 3: Prophylactic surgery among patients with high-penetrance mutations
(BRCA1 and BRCA2 founder mutations) and low-penetrance mutations (APC I1307K).
Characteristic
BRCA1
185delAG
BRCA1
5382insC
BRCA2
6174delT
BRCA1 del
exons 9-12
APC I1307K
N 3 1 3 3 16
Age
Mean (SD) 54.3 (17.21) 41 (0) 52 (9.54) 36 (7) 47.8 (17.41)
Median 47 41 53 33 48.5
Range (42, 74) (41, 41) (42, 61) (31, 44) (25, 79)
Gender
Female 1 (33.3) 1(100) 2 (66.7) 3 (100) 11 (68.8)
Male 2 (66.7) 0 1 (33.3) 0 5 (31.3)
Ethnicity
Hispanic 1 (33.3) 0 0 3 (100) 1(6.3)
Non Hispanic, White 2 (66.7 1 (100) 3 (100) 0 15 (93.8)
Personal Cancer
History

Affected 2 (66.7) 1 (100) 0 2(66.7) 11 (68.8)
Not Affected 1 (33.3) 0 3 (100) 1(33.3) 5 (31.3)
Surgery Post Genetic Testing
Mastectomy 0 1
c
0 2
e,f
0
h

Cancer 0 1 0 0 N/A
Prevention 0 1 0 2 N/A
Hysterectomy
0 0 1 2 0
h

Cancer N/A
a
N/A 0 2
f,g
N/A
Prevention N/A N/A 1 0 N/A
Oophorectomy
1

1

2

2 0
h

Cancer 0 0 0 2
f,g
N/A
Prevention 1
b
1
c
2
d
0 N/A
MICRA: Distress (0-30)
Mean (SD) 10 (0) 14 (0) 2.5 (0.71) 10.3 (5.51) 2.6 (4.83)
Median 10 14 2.5 13 0.5
Range (10, 10) (14, 14) (2, 3) (4, 14) (0, 18)
MICRA: Uncertainty (0-45)
Mean (SD) 17 (0) 16 (0) 10.5 (0.71) 17.8 (17.76) 6.9 (9.04)
Median 17 16 10.5 8 2
Range (17, 17) (16, 16) (10, 11) (7, 38.25) (0, 26)
MICRA: Positive (0-20)
Mean (SD) 2 (0) 7 (0) 6.5 (2.12) 12.7 (5.03) 9.6 (6.38)
Median 2 7 6.5 12 8.5
Range (2, 2) (7, 7) (5, 8) (8, 18) (0, 20)

a
No patients with BRCA1 185delAG were under consideration for prophylactic
hysterectomy due to gender or a history of cancer in the relevant organ.
b
One female patient with BRCA1 185delAG was diagnosed with left breast cancer in
early 2016 and underwent neoadjuvant chemotherapy and lumpectomy. She came in
for genetic testing in March 2016, revealing a BRCA1 mutation. She subsequently
underwent a preventive bilateral salpingo-oophorectomy (BSO) in later May 2016.
25
c
One patient was diagnosed with triple negative breast cancer of the right breast in
June 2015. After finding out she was a BRCA1 carrier, she underwent bilateral
mastectomy (treatment for cancer in the right breast and prevention in the left breast)
and a BSO in November 2015.
d
One female patient underwent genetic testing for family history of ovarian cancer. She
was found to have a BRCA2 mutation and underwent a preventive BSO in December
2014. Another female patient underwent a total abdominal hysterectomy and bilateral
salpingo-oophorectomy (TAH-BSO) in her youth due to her family history. She
underwent genetic testing in 2016 and was found to carry a BRCA2 mutation. She is in
the process of considering a preventive mastectomy.
e
One female patient was unaffected with cancer and is undergoing evaluation for a
preventive bilateral mastectomy.
f
One female patient was affected with ovarian cancer and underwent a TAH-BSO to
treat her cancer and then subsequently underwent a preventive bilateral mastectomy
one year later.
g
One female patient was affected with ovarian cancer and metastatic ovarian cancer.
She underwent a TAH-BSO. However, the patient is undergoing chemotherapy for
metastatic lesions to the brain and lung.
h
No patients with APC I1307K were under consideration for prophylactic mastectomy,
hysterectomy, or oophorectomy due to gender or a history of cancer in the relevant
organ.

26
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Creator Idos, Gregory (author)

Core Title A multi-center prospective cohort study of the diagnostic yield and patient experience of multiplex gene panel testing for hereditary cancer risk

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 12/03/2019

Defense Date 11/20/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cancer susceptiblity,genetic testing,germline,hereditary risk,multigene panel,OAI-PMH Harvest,pathogenic variants

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Advisor Gruber, Stephen (committee chair), Buxbaum, James (committee member), Lenz, Heinz-Josef (committee member)

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