Articles

Blood-based tests for multicancer early detection

(PATHFINDER): a prospective cohort study
Deb Schrag, Tomasz M Beer, Charles H McDonnell III, Lincoln Nadauld, Christina A Dilaveri, Robert Reid, Catherine R Marinac, Karen C Chung,
Margarita Lopatin, Eric T Fung, Eric A Klein

Summary
Background Multicancer early detection (MCED) blood tests can detect a cancer signal from circulating cell-free DNA Lancet 2023; 402: 1251–60
(cfDNA). PATHFINDER was a prospective cohort study investigating the feasibility of MCED testing for cancer See Comment page 1213
screening. Memorial Sloan Kettering
Cancer Center, New York, NY,
USA (Prof D Schrag MD); Exact
Methods In this prospective cohort study done in oncology and primary care outpatient clinics at seven US health
Sciences, Madison, WI, USA
networks, a convenience sample of adults aged 50 years or older without signs or symptoms of cancer consented to (Prof T M Beer MD); Sutter
MCED testing. We collected blood, analysed cfDNA, and returned results to participants’ doctors. If a methylation Health, Sacramento, CA, USA
signature indicative of cancer was detected, predicted cancer signal origin(s) informed diagnostic assessment. The (C H McDonnell III MD);
Intermountain Healthcare,
primary outcome was time to, and extent of, diagnostic testing required to confirm the presence or absence of cancer.
St George, UT, USA
This trial is registered at ClinicalTrials.gov, NCT04241796, and is completed. (L Nadauld MD); Mayo Clinic,
Rochester, MN, USA
Findings Between Dec 12, 2019, and Dec 4, 2020, we recruited 6662 participants. 4204 (63·5%) of 6621 participants (C A Dilaveri MD); US Oncology
Research, VA Cancer Specialists,
with analysable results were women, 2417 (36·5%) were men, and 6071 (91·7%) were White. A cancer signal was
Fairfax, VA, USA (R Reid MD);
detected in 92 (1·4%) of 6621 participants with analysable results. 35 (38%) participants were diagnosed with cancer Dana-Farber Cancer Institute,
(true positives) and 57 (62%) had no cancer diagnosis (false positives). Excluding two participants whose diagnostic Boston, MA, USA
assessments began before MCED test results were reported, median time to diagnostic resolution was 79 days (C R Marinac PhD); GRAIL,
Menlo Park, CA, USA
(IQR 37–219): 57 days (33–143) in true-positive and 162 days (44–248) in false-positive participants. Most participants
(K C Chung MS,
had both laboratory tests (26 [79%] of 33 with true-positive results and 50 [88%] of 57 with false-positive results) and E T Fung MD PhD,
imaging (30 [91%] of 33 with true-positive results and 53 [93%] of 57 with false-positive results). Fewer procedures Prof E A Klein MD, M Lopatin MS)
were done in participants with false-positive results (17 [30%] of 57) than true-positive results (27 [82%] of 33) and Correspondence to:
few had surgery (one with a false-positive result and three with a true-positive result). Prof Deb Schrag, Memorial Sloan
Kettering Cancer Center,
New York, NY 10065, USA
Interpretation This study supports the feasibility of MCED screening for cancer and underscores the need for further schragd@mskcc.org
research investigating the test’s clinical utility.

Funding GRAIL.

Copyright © 2023 Elsevier Ltd. All rights reserved.

Introduction
Cancer is the second most common cause of death in
Europe and the USA and, globally, more than 1·9 million
new cases are expected to be diagnosed in 2023.1
Recommended cancer screening tests have reduced
cancer-related mortality, but are only available for a few
cancer types and represent less than 25% of cancer
deaths in the USA.2 The US Preventive Services Task
Force advises screening (grade A/B) for breast, lung,
colorectal, and cervical cancers. For many other cancer
types, screening strategies either lack well established
net benefit or do not exist. As a result, many cancers are
identified at late stages, when treatment is less
effective.3
Advances in genomics and machine learning have
enabled development of blood tests for cancer screening,
monitoring, and treatment selection.4–10 Multicancer early
detection (MCED) blood tests have the potential to
improve the efficiency and convenience of screening and
do not carry the risks of whole-body imaging. However,
these tests are new and insight about how doctors and
patients respond to a positive MCED test is limited.7–10
The PATHFINDER study was designed to address this
knowledge gap.
The MCED test investigated in this study detects
cancer-specific DNA methylation patterns from cell-free
DNA (cfDNA) shed by tumours into circulating blood. If
a cancer signal is detected, the test also predicts the
cancer signal origin (CSO; ie, the cancer or tissue type
where the tumour arose).7,8 This test was developed and
validated in the Circulating Cell-Free Genome Atlas
(CCGA) study (NCT02889978, US Food and Drug
Administration Investigational Device Exemptions
G190251). In the CCGA study, the test detected a
methylation-based signal for more than 50 distinct
cancer types from a single blood draw and accurately
predicted CSO in 1273 (89%) of 1435 participants.7,8 We
aimed to investigate the feasibility of MCED testing in
outpatient settings for adults older than 50 years without
symptoms of cancer.

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Research in context
Evidence before this study
Previous evidence has established that cancers shed cell-free DNA
(cfDNA) into the bloodstream that is detectable before the onset
of clinical signs and symptoms, creating a window of preclinical
detectability. We searched PubMed using the terms “multicancer
early detection” OR “multicancer detection” OR “liquid biopsy
AND cancer screening” for publications in English published
between Jan 1, 1991, and July 29, 2023, and found 2415 results.
After excluding non-human studies, reviews, commentaries,
health economics-focused articles, and single-cancer studies,
we identified 12 analytic or case-control studies using various
technologies to detect cancer signals of multiple tumour types in
blood. Collectively, these studies have established the feasibility
and putative performance characteristics of genomic-based tools
to detect cancers and predict their tissue of origin that could serve
to establish a new blood-based screening framework. To date,
one published cohort study has investigated the feasibility and
safety of a blood-based assay for multicancer detection, but this
study was limited to women in a single health system and relied
on whole-body imaging rather than a blood test for tumour
localisation. Little evidence exists about the diagnostic
assessments that ensue following the use of this technology, the
test performance characteristics, or how patients and clinicians
deal with the results.
Methods
Study design and participants
In the PATHFINDER study, we assessed use of MCED
testing in a prospective cohort study done in oncology and
primary care outpatient clinics at seven US health
networks, including hospitals and community-based
clinics. Eligible participants, a non-consecutive
convenience sample, consented to MCED testing and
1 year of follow-up surveillance of their electronic health
records. No specific diagnostic or screening assessments
were mandated. All study procedures were approved by a
central or local institutional review board. Additional
details regarding study design have been previously
published.11
Eligible participants were adults aged 50 years or older,
with or without additional cancer risk factors. Participants
with additional risk met at least one of the following
criteria: a history of smoking at least 100 cigarettes within
the participant’s lifetime, cancer predisposition based on
meeting criteria for germline testing from the National
Comprehensive Cancer Network guidelines12 or having
known hereditary cancer syndrome, or personal history
of cancer with definitive treatment completed at least
3 years before enrolment.11 Participants were excluded if
they were undergoing diagnostic assessment for
suspicion of cancer or cancer recurrence, had a history of
untreated cancer, or had been treated for cancer within
3 years of enrolment. Participants provided written
informed consent.
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Added value of this study
PATHFINDER assessed clinician-guided diagnostic
assessments for adults who received positive results from a
targeted-methylation blood-based multicancer early
detection (MCED) test as well as the extent of imaging and
diagnostic testing needed to reach diagnostic resolution.
The study measured performance characteristics of this test
applied among adults aged 50 years or older with and
without increased cancer risk seen in outpatient medical
practice. These findings add to emerging evidence supporting
the feasibility of MCED testing to screen for multiple types of
cancer simultaneously from a single blood specimen and
characterise the diagnostic pathways that follow receipt of a
positive MCED test.
Implications of all the available evidence
These results support further development of MCED testing
for cancer screening and provide preliminary estimates of the
consequences and diagnostic yield of this emerging
technology. Further research is needed to investigate the
clinical utility of MCED testing to screen for cancer in diverse
populations.
Procedures
Participants contributed a blood specimen, obtained at
outpatient ambulatory care clinics, typically by
phlebotomists. Plasma isolation, followed by cfDNA
extraction, was done as previously described.7,8,11 Results
of the MCED test were returned to the ordering doctor
within approximately 15 days and then shared with
participants. The MCED test report provided a binary
result (cancer signal detected or not detected), including
prediction of the most likely CSOs if a signal was
detected. If a cancer signal was detected, participants had
diagnostic assessments coordinated by, and at the
discretion of, their doctor. Doctors determined when the
diagnostic work-up was considered complete. Parameters
for this diagnostic assessment were not stipulated by the
protocol, and the study sponsor supported all costs. This
design enabled insight into the diagnostic pathways
followed in response to MCED test results and CSO
predictions. All participants, including those with no
cancer signal detected, were advised to continue usual
medical care and guideline-recommended cancer
screening.
We did an end-of-study cancer-status assessment
12 months after enrolment, which included a review of
electronic health records to confirm the presence or
absence of cancer. Record review was supplemented with
telephone contact as needed. Cancer status assessment
was considered complete if a cancer diagnosis was
reported at any time during the follow-up period or there
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was no cancer diagnosis recorded in the electronic health
records or through patient contact 12 months after the
MCED blood draw.
The MCED test used in PATHFINDER uses next-
generation sequencing to interrogate cfDNA in peripheral
blood to measure the extent and location of genomic
DNA methylation patterns, which are characteristic of
cancers13 and specific to cancer type. A computational
algorithm identifies whether a cancer signal is present
and, if so, a second algorithm predicts the most likely
tumour type, labelled the CSO. This prediction might
help to direct diagnostic assessment.8,14 We assigned CSO
predictions from a set of 21 predetermined categories that
correspond to major tumour types described (appendix
p 5),7,8,11 and are ranked in order of likelihood. We
calculated CSO prediction accuracy as the number of
patients with a correctly predicted tumour type among all
patients with a confirmed cancer diagnosis following a
positive MCED assay. Technical aspects of the assay and
clinical validation have been described previously.7,8
Outcomes
The primary objective was to investigate the time
required to achieve diagnostic resolution following
receipt of a positive MCED test and to assess the extent of
testing pursued. Time to diagnostic resolution for each
participant was defined as the number of days between
the date that test results were available to the ordering
doctor through the reporting portal and the date of
diagnostic resolution as determined by the ordering
doctor.
For each participant, the extent of diagnostic testing
included all clinic visits, laboratory and imaging tests,
and procedures recorded between the date a positive test
result was available in the portal and the date of diagnostic
resolution or end of study, whichever came first.
Procedures were categorised as either non-surgical
(eg, endoscopy, bone marrow biopsy, core biopsy, fine
needle aspiration, and pap smear) or surgical.
Cancer diagnosis
A cancer diagnosis was established by pathological,
laboratory, or radiographic confirmation. 113 (93%) of
122 cancers were pathologically confirmed. Malignancies
were categorised as either invasive solid tumours
(excluding non-metastatic basal cell carcinoma and
squamous cell carcinoma of the skin) or haematological.
Cancer diagnosis and staging for solid tumours was
based on the American Joint Committee on Cancer
(AJCC) Staging Manual (version 8) and on AJCC or the
Ann Arbor staging systems for haematological
malignancies.15 For participants with a cancer history of
the same tumour type identified from MCED testing,
staging was categorised as either recurrent locoregional
or metastatic for solid tumours and as recurrent for
haematological malignancies. Premalignant conditions
(eg, cervical dysplasia or monoclonal gammopathy of
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Articles
undetermined significance [MGUS]) were not considered
cancer.
A 12-month follow-up period has traditionally been used
to assess the performance characteristics of single cancer-
screening modalities, including mammography,16–18 low-
dose CT screening for lung cancer19–21 and ovarian cancer
screening.22 The same interval was chosen for this study to
facilitate assessment of MCED test performance compared
with these other cancer screening tests. This arbitrary
reference was not informed by biological considerations.
Within 12 months of enrolment, participants with an
MCED cancer signal detected and a clinically confirmed
cancer were classified as true positive and those without
confirmed cancer as false positive. Participants with no See Online for appendix
MCED cancer signal detected but who had a confirmed
cancer diagnosis within 12 months were classified as
false negative. False negatives were defined as those
participants with cancers detected by means other than
the diagnostic work-up prompted by a positive MCED
test, which included those detected by routine screening
assessments that occurred during the 12-month study
period or those detected because the patient developed
signs or symptoms. Screening was defined as standard if
it was consistent with US Preventive Services Task Force
(USPSTF) grade A, B, or C recommendations (breast,
colorectal, cervical, prostate, or high-risk lung), although
not all institutions follow USPSTF guidelines
specifically.23 Cancers detected by non-standard screening
included cancers of the ovary, testis, thyroid, and skin
without USPSTF recommendations. Clinically detected
cancers were defined as those diagnosed on the basis of a
patient’s presentation with clinical signs or symptoms
such as pain, unexplained weight loss, or a palpable
mass. More detail on the definitions of cancers detected
by standard screening, cancers detected by non-standard
screening, and clinically detected cancers is provided in
the appendix (p 30).
Safety
Safety assessments included a summary of adverse
events reported and defined by site investigators at any
time during the study, including at phlebotomy, results
disclosure, or diagnostic assessments in those with a
cancer signal detected. Adverse events were reported to
the study sponsor and the relevant institutional review
board or independent ethics committee. An independent
Data Safety Monitoring Board provided oversight of the
study conduct.
Test performance
As a secondary objective, MCED test performance was
assessed with the following measures: resolution (true
positive or false positive), positive and negative predictive
value, specificity, and CSO accuracy. Participants with an
indeterminate CSO were excluded from CSO accuracy
calculations. Additional performance endpoints were the
cancer yield rate defined as the number of true positives
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among all participants tested and the number needed to
screen to detect one cancer.
We collected participant-reported outcomes relating to
anxiety, distress, uncertainty, and MCED test satisfaction
at the time of initial blood draw, when test results were
returned, when diagnostic resolution was achieved, and
at the end of the study, and these will be reported in a
separate manuscript. We did a post hoc exploratory
analysis of circulating tumour allele fraction (cTAF) for
all cancers detected during the study. The detection
threshold was defined as 3·1 × 10–⁴ cTAF at
99·3% specificity.24 All outcome measures are defined in
the appendix (p 6).
Statistical analysis
Analyses were descriptive with no pre-specified
hypothesis testing. The analysis plan assumed that
70% of enrolled participants would have at least one
additional cancer risk factor. Simulations estimated that
a target enrolment of 6200 participants would generate
106–141 cancer signals detected.11 The primary analysis
was based on participants with a diagnostic assessment
triggered by a cancer signal detected. Two-sided
Wilcoxon 95% CIs are reported for all test performance
measures. Analyses were done using R (version 4.1.2).
Protocol deviations occurred in 106 (1·6%) of 6662
participants, including important protocol deviations
Outcome analysis sets—true positives
36 cancers
1 true positive had 2 cancers
35 true positives
2 true positives began 1 true positive had
diagnostic workup indeterminate CSO
before return of
MCED test results
33 evaluated for extent of 34 assessed for CSO
diagnostic testing prediction accuracy
92 cancer signal detected
35 true positive
Figure 1: Trial profile
Participant flow diagram, including true-positive analysis sets for outcomes with variable participant numbers. CSO=cancer signal origin. MCED=multicancer early
detection.
1254
in 55 (0·8%; appendix p 7). This study is registered with
ClinicalTrials.gov, NCT04241796.
Development of a refined MCED test
After PATHFINDER was launched, a refined MCED test
version was developed, using a larger CCGA training
dataset. All elements of this refined test were locked
before sample processing, validation was done in a new
independent test set, and the statistical analysis plan was
finalised before evaluation. MCED test refinements
included: (1) an increased cancer signal detection
threshold for haematological signals to reduce false
positives from non-malignant hematological conditions
such as MGUS, and decreased threshold for solid tumour
signals to increase the detection of solid cancers;
(2) elimination of the “indeterminate” label for CSO to
generate a prediction for all test-positive samples; and
(3) restriction of CSO predictions to the two most likely
tumour types. Results of testing using the refined MCED
test were not returned to either doctors or participants,
and therefore did not influence diagnostic assessments.
Role of the funding source
The study sponsor contributed to the study design, data
analysis, interpretation, and drafting, review, and
approval of the manuscript. The sponsor used a medical
writing agency to assist with manuscript preparation.
6662 participants enrolled
32 retrospectively ineligible
6630 clinically eligible
5 withdrew consent
6625 clinically evaluable
4 assay result not evaluable
6621 with evaluable MCED results
6529 no cancer signal detected
57 false positive 6235 true negative 86 false negative 208 no end-of-study
assessment
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Results
Aged ≥50 years Aged ≥50 years Total
Between Dec 12, 2019, and Dec 4, 2020, we enrolled with additional without additional (n=6621)
6662 participants, 6625 (99·4%) of whom were risk (n=3681) risk (n=2940)
clinically evaluable, and 6621 (99·4%) had analysable Age, years* 64·0 (58·0–71·0) 61·0 (55·0–67·0) 63·0 (56·0–70·0)
MCED results (figure 1). Of these 6621 participants, Age group, years
3681 (55·6%) were considered to have additional risk 50–64 1858 (50·5%) 1933 (65·7%) 3791 (57·3%)
factors. Participants were predominately White 65–79 1637 (44·5%) 931 (31·7%) 2568 (38·8%)
(6071 [91·7%]) and highly educated (4276 [64·6%] with ≥80 186 (5·1%) 76 (2·6%) 262 (4·0%)
college degrees), and a smaller proportion were current
Sex
smokers (268 [4·0%]) compared with the general
Female 2393 (65·0%) 1811 (61·6%) 4204 (63·5%)
population (table 1). 1622 (24·5%) participants had a
Male 1288 (35·0%) 1129 (38·4%) 2417 (36·5%)
previous cancer history; demographic details and
Race or ethnicity
MCED test performance characteristics in this subset
Asian† 39 (1·1%) 90 (3·1%) 129 (1·9%)
are given in the appendix (p 8). At enrolment, most
Hispanic 66 (1·8%) 68 (2·3%) 134 (2·0%)
participants were up to date with cancer screening,
Non-Hispanic Black 44 (1·2%) 46 (1·6%) 90 (1·4%)
with 4492 (91·9%) of 4888 reporting recent colorectal
Non-Hispanic White 3441 (93·5%) 2630 (89·5%) 6071 (91·7%)
cancer screening and 2854 (80·5%) of 3547 women
Other‡ 28 (0·7%) 38 (1·3%) 66 (1·0%)
reporting up-to-date breast cancer screening (according
Missing 63 (1·7%) 68 (2·3%) 131 (2·0%)
to USPSTF guidelines) among eligible participants
BMI, kg/m²
with complete data (table 1).
A cancer signal was detected in 92 (1·4%) of <18·5 32 (0·9%) 18 (0·6%) 50 (0·8%)

6621 participants (figure 1), with 56 (1·5%) of 3681 in 18·5 to <25·0 1045 (28·4%) 956 (32·5%) 2001 (30·2%)
those with additional risk and 36 (1·2%) of 2940 in those 25·0 to <30·0 1297 (35·2%) 1039 (35·3%) 2336 (35·3%)
without additional risk (table 2). Of those with a cancer ≥30·0 1264 (34·3%) 887 (30·2%) 2151 (32·5%)
signal detected, 35 (38%) of 92 were diagnosed with Other or missing 43 (1·2%) 40 (1·4%) 83 (1·3%)
cancer (true positives) and 57 (62%) had no cancer Education
diagnosis (false positives; figure 1). Most true positives Less than high school 50 (1·4%) 15 (0·5%) 65 (1·0%)
(27 [77%] of 35) had a cTAF above 3·1 × 10–⁴, the MCED High school graduate 345 (9·4%) 150 (5·1%) 495 (7·5%)
clinical detection threshold (appendix p 31). The number Some college 1060 (28·8%) 645 (21·9%) 1705 (25·8%)
of positive MCED tests (n=92) was lower than the College graduate 2176 (59·1%) 2100 (71·4%) 4276 (64·6%)
projected 106–141 in the sample size calculations. Other or missing 50 (1·4%) 30 (1·0%) 80 (1·2%)
Of those without a cancer signal detected, Smoking status
6235 (95·5%) of 6529 were true negatives, 86 (1·3%) were Current smoker 268 (7·3%) 0 268 (4·0%)
false negatives, and 208 (3·2%) did not have a cancer- Former smoker 2229 (60·6%) 0 2229 (33·7%)
status assessment at the end of the study. 82 (95%) of Non-smoker 1184 (32·3%) 2940 (100%) 4124 (62·3%)
86 false negatives had a cTAF below 3·1 × 10–⁴ (appendix Eligible for lung cancer screening§ 223 (6·1%) 0 223 (3·4%)
p 31). Previous cancer history 1622 (44·1%) 0 1622 (24·5%)
Of the 92 participants with a cancer signal detected, Cancer predisposition 425 (11·5%) 0 425 (6·4%)
two (2%) began their diagnostic assessment because of Up to date with standard cancer screening before MCED testing
symptoms or physical examination finding after the Colorectal cancer¶ 2404/2628 (91·5%) 2088/2260 (92·4%) 4492/4888 (91·9%)
blood draw and before the MCED test results were Breast cancer|| 1504/1930 (77·9%) 1350/1617 (83·5%) 2854/3547 (80·5%)
returned and are not included in the analysis of the
Data are n (%), n/N (%), or median (IQR). Total includes all clinically evaluable patients with analysable MCED results.
extent of diagnostic testing (appendix p 10). Median MCED=multicancer early detection. USPSTF=US Preventive Services Task Force. *Age was truncated at 85 years to
numbers of clinic visits, laboratory tests, and imaging protect confidentiality. †Non-Hispanic Asian, Native Hawaiian, or Pacific Islander. ‡Includes unknown,
tests were similar for true-positive and false-positive American Indian, or Alaska Native. §Satisfy approved USPSTF criteria for lung cancer screening using low-dose CT.
¶Participants aged 75 years or younger, up to date with USPSTF colorectal cancer screening recommendations
participants, whereas true positives were more likely (n=4888 eligible with complete information). ||Women aged 50–74 years up to date with breast cancer screening
than false positives to undergo both non-surgical and (USPSTF recommendations, MRI, or ultrasound; n=3547 eligible with complete information).
surgical procedures (appendix p 10).
Table 1: PATHFINDER participant demographics and baseline characteristics
Median observed time to diagnostic resolution was
79 days (IQR 37–219). True positives had a shorter
median time to resolution (57 days [33–143]) compared count with differential (48 [53%]), and protein tumour
with false positives (162 days [44–248]; figure 2). Most markers (39 [43%]; appendix p 10).
true positives (24 [73%] of 33) had diagnostic resolution At least one imaging test was done in
within 3 months (figure 2). 83 (92%) of 90 participants with a cancer signal detected
76 of 90 participants with positive MCED results had (figure 3), and 44 (53%) of those had more than one
laboratory tests (figure 3). The most common laboratory imaging study. PET-CT (55 [61%] of 90), CT (35 [39%]),
tests were a metabolic panel (51 [57%]), complete blood and MRI (19 [21%]) were the most common imaging

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Resolution
All
True positive
False positive
Positive predictive value
Negative predictive value
Specificity
Yield rate
Number needed to screen
Predicted origin accuracy*
First CSO correct
First or second CSO correct
Data are n (%), n/N, or % (95% CI). CSO=cancer signal origin. *Excludes one participant with indeterminate CSO from the true-positive set.
Table 2: Multicancer early detection test performance
tests (appendix p 10). Diagnostic tests done in fewer than
three participants are listed in the appendix (p 22).
32 (73%) of 44 participants with a haematological CSO
had a PET-CT scan as their first imaging test, consistent
with diagnostic guidelines.25
Of the 90 participants with a diagnostic assessment
triggered by MCED testing, 44 (49%) of 90 had at least
one procedure (figure 3; appendix p 10). More true-
positive than false-positive participants had procedures
(figure 3). In most false-positive participants, procedures
were prompted by abnormal imaging. Three true-positive
participants had surgical procedures (lymph node
mesenteric excision, mesenteric mass and mesenteric
lymph node biopsy, and base of tongue biopsy). Only
one participant with a false-positive result had a surgical
procedure (inguinal orchiectomy), prompted by
abnormal imaging.
Within 12 months after enrolment, 122 cancers were
diagnosed in 121 participants (appendix p 14). Of these,
35 (29%) had a cancer signal detected by MCED,
38 (31%) were detected through screening (29 in
accordance with USPSTF guidelines and nine by non-
standard screening tests; appendix p 32) and the remaining
48 (40%) were clinically detected. Of the 35 true positives,
24 (69%) of 35 were in the additional-risk cohort;
28 (80%) of 35 had a new cancer, six (17%) had recurrent
cancer, and one (3%) had both, for a total of 36 cancers
(19 [53%] solid tumours and 17 [47%] haematological
malignancies (figure 4). In true positives with a new cancer
diagnosis, 14 (48%) of 29 cancers were stage I–II (figure 4;
appendix p 14): six solid tumours and eight haematological
malignancies. MCED identified a tumour type that does
not have a USPSTF screening recommendation in
26 (74%) of 35 participants (figure 4; appendix p 14). Most
new cancers diagnosed in false negatives were stage I–II
(55 [73%] of 75; appendix p 16). Recurrent cancers were
identified in 11 (13%) of 86 participants with false-negative
results. The histology results for cancers diagnosed in
1256
Age ≥50 years with additional Age ≥50 years without Total
cancer risk (n=3681) additional cancer risk (n=2940) (n=6621)
56 (1·5%) 36 (1·2%) 92 (1.4%)
24 (0·7%) 11 (0·4%) 35 (0·5%)
32 (0·9%) 25 (0·9%) 57 (0·9%)
24/56; 43% (30·8–55·9) 11/36; 31% (18·0–46·9) 35/92; 38% (28·8–48·3)
3449/3502; 98·5% (98·0–98·8) 2786/2819; 98·8% (98·4–99·2) 6235/6321; 98·6% (98·3–98·9)
3449/3480; 99·1% (98·7–99·4) 2786/2810; 99·1% (98·7–99·4) 6235/6290; 99·1% (98·9–99·3)
24/3681; 0·65% (0·41–0·92) 11/2940; 0·37% (0·17–0·61) 35/6621; 0·53% (0·36–0·71)
3681/24; 153 (108–245) 2940/11; 267 (163–588) 6621/35; 189 (141–276)
20/23; 87% (67·9–95·5) 9/11; 82% (52·3–94·9) 29/34; 85% (69·9–93·6)
23/23; 100% (85·7–100) 10/11; 91% (62·3–99·5) 33/34; 97% (85·1–99·8)
those with false-negative MCED results are shown in the
appendix (p 18).
Overall positive predictive value was 38% (35/92) and
was higher in those with additional risk (24 [43%] of 56)
and lower in those without additional risk (11 [31%] of 36;
table 2). Negative predictive value was 98·6% (6235/6321;
table 2). Specificity was 99·1% (6235/6290). The cancer
yield rate was 0·53% (35/6621), corresponding to a
number needed to screen to detect one cancer of 189.
A correct first or second CSO prediction was returned
for 33 (97%) of 34 true positives, excluding one participant
with an indeterminate CSO (table 2). The one participant
with an inaccurate first (colon or rectum) and second
(upper gastrointestinal tract) CSO prediction was
diagnosed with adenocarcinoma of the small intestine. Of
the 57 false positives, 35 (61%) had a haematological CSO
prediction; of these, 12 (34%) had a haematological
precursor condition, such as MGUS (appendix p 23).
Adverse events were reported for four participants,
two with events related to phlebotomy (anxiety and
bruising at the venipuncture site) and two with anxiety
reported before the MCED test results were returned. All
events were mild in severity.
Results of the refined MCED test are given in
the appendix (pp 25, 27, 28, 34). 6578 (98·7%) of
6662 participants had analysable results for the refined
MCED test. The refined test version improved
specificity (99·5% [95% CI 99·3–99·6]) and had a
positive predictive value (43·1% [31·2–55·9]) similar to
that of the study test version (table 2; appendix p 27).
Characteristics of cancers detected with the refined
MCED test are summarised in the appendix (p 28).
Concordance between the study and refined versions of
the MCED test is shown in the appendix (p 34). The
refined MCED test reduced the proportion of participants
with a haematological CSO prediction among false
positives from 35 (61%) of 57 to four (12%) of 33. With
the refined MCED test version, three of the
www.thelancet.com Vol 402 October 7, 2023

Median 57 days
(IQR 33–143)
73% (24/33) <3 months
True positive
Median 162 days
(IQR 44–248)
42% (24/57) <3 months
False positive
0 100 200
Days to diagnostic resolution
Figure 2: Time to diagnostic resolution for participants with cancer signal
detected (n=90)
four participants with false-positive results predicted to
have a haematological malignancy had a precursor
condition such as MGUS.
Discussion
The PATHFINDER study provides early evidence of the
feasibility of blood testing to screen for multiple cancers
with a single test, a departure from the prevailing
framework requiring distinct screening tests for each
cancer type. When the MCED assay was positive and
cancer was present, the test accurately predicted the
tumour origin, and half of all cancer diagnoses were
made in less than 2 months. When cancer could not be
confirmed, diagnostic assessments, often with serial
scans, continued for a longer duration, typically extending
for 4 months. Blood tests and advanced imaging studies
were commonly used following a signal detected result.
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Articles
True positive False positive
Any laboratory test 26/33 (79%) 50/57 (88%)
Any imaging test 30/33 (91%) 53/57 (93%)
Any procedure 27/33 (82%) 17/57 (30%)
Any non-surgical procedure 26/33 (79%) 16/57 (28%)
Any surgical procedure 3/33 (9%) 1/57 (2%)
100 50 0 50 100
Participants (%)
Figure 3: Extent of diagnostic testing in participants with cancer signal detected (n=90)
Importantly, the MCED assay detected many cancer
types for which screening tests do not exist, including
some found at early stages. For example, cancers of the
bile duct, small intestine, pancreas, and a spindle-cell
neoplasm were detected at early stages amenable to
surgical resection. These lethal malignancies are
unlikely to have been identified upon routine physical
examination or screening. Haematological malignancies,
particularly follicular lymphomas, were also detected at
early stages, although the clinical significance of these
more indolent neoplasms is less certain. Nearly half of
the newly diagnosed cancers were identified at an early
stage.
At the study’s inception, participants were highly
adherent to established cancer screening and some
300 400
received forms of screening that extend beyond USPSTF
recommendations. This healthy volunteer effect might
help explain why the number of cancers diagnosed by
MCED testing was less than expected. The lower-than-
projected rate of enrolment of individuals with cancer
risk factors and uncertainty in assumptions regarding
cancer dwell times might also explain the lower-than-
expected rate of cancer detection.
Because this study represented a prevalence round of
screening, indolent haematological conditions were
likely to have been overrepresented compared with what
would be detected in later rounds of repeated screening.
The high rate of screening participation in the study
population might also explain both the number and
types of cancers diagnosed in the absence of an MCED
signal. For example, eight of the 11 total lung cancers
diagnosed in this study arose in individuals who did not
meet USPSTF 2021 criteria for low-dose CT screening.26
Most of these lung cancers were early stage, and all had
cTAF below the MCED detection threshold. By contrast,
the one case of lung cancer detected by MCED testing
presented at stage III.
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Oropharyngeal cancer (n=2)
Squamous cell carcinoma stage II
Squamous cell carcinoma stage IV
Lung (n=1)
Adenocarcinoma stage III
Breast (n=5)
Carcinoma consistent with breast primary RM
Carcinoma consistent with breast primary RM
Carcinoma consistent with breast primary RM
Carcinoma consistent with breast primary RM
Carcinoma consistent with breast primary RM
Liver (n=1)
No pathology stage I
Intrahepatic bile ducts (n=1)
Adenocarcinoma stage III
Pancreas (n=1)
Adenocarcinoma stage II
Small intestine (n=1)
Adenocarcinoma stage I
Colon and rectum (n=2)
Adenocarcinoma stage IV
No pathology stage IV
Uterus (n=1)
Endometrial adenocarcinoma stage I
Ovary (n=1)
Serous adenocarcinoma stage III
Of the 29 participants with new cancers,
Prostate (n=2)
14 (48%) were stage I or II
Adenocarcinoma stage IV
Biochemical recurrence, no pathology
Bone (n=1)
Spindle-cell neoplasm stage II
Figure 4: Cancer diagnosed after a positive multicancer early detection result
The 36 cancers diagnosed in the 35 true-positive participants are depicted by a dot in the organ or system in which the cancer was found. Histology details are given
when available. Overall, 26 (74%) of 35 participants had cancers diagnosed that did not have USPSTF recommendations for screening tests. RM=recurrent metastatic
cancer. USPSTF=US Preventive Services Task Force.
The distribution of cancers in participants with a
false-negative result did not have a clear pattern, other
than the predictable outcome of being enriched for
cancers that are commonly screen-detected such as
breast and colorectal cancers. Most false negatives had
cTAF below the MCED test limit of detection.24,27–29
Because MCED testing technologies are still in early
phases of development, rely on different approaches to
cohort ascertainment, and do not have a consistent
gold-standard definition of a true positive, it is infeasible
to compare the sensitivity of different MCED test
methods.
False-positive results and the potential for
overdiagnosis are inherent risks of all screening
methods. In this study, fewer than 1% of participants
had false-positive results, which compares favourably
with recommended single-cancer screening tests.18,30,31 In
the CCGA study,7,32 we found that cancers with detectable
circulating tumour DNA were more likely to be lethal
than those with undetectable levels of circulating tumour
DNA. This finding suggests that MCED testing on the
basis of circulating tumour DNA technology might
minimise the risk of overdiagnosis by preferentially
detecting more lethal tumours. However, larger trials
with longer-term follow-up are needed to quantify rates
of overdiagnosis.
1258
Lymphoma (n=12)
Follicular lymphoma stage I
Follicular lymphoma stage I
Marginal-zone lymphoma stage I
Hodgkin lymphoma stage I
Follicular lymphoma stage II
Follicular lymphoma stage II
Follicular lymphoma stage II
Follicular lymphoma stage II
Diffuse large B-cell lymphoma stage III
Mature B-cell neoplasm stage IV
Diffuse large B-cell lymphoma stage IV
Recurrent local cancer, no pathology
Waldenstrom macroglobulinaemia (n=2)
Waldenstrom macroglobulinaemia
Waldenstrom macroglobulinaemia
Lymphoid leukaemia (n=2)
Chronic B-cell lymphocytic leukaemia
Chronic B-cell lymphocytic leukaemia
Plasma-cell myeloma (n=1)
Plasma-cell neoplasm
USPSTF screening
No USPSTF screening
Study strengths include usage of a validated fixed
assay and algorithm to predict both cancer signal
presence and CSO, and recruitment of participants
from various health-care settings in US ambulatory care
practice. The absence of protocol-mandated diagnostic
algorithms allowed insight into clinician judgement
regarding clinical management of a positive MCED test
result.
The study had several limitations. First, access to
advanced imaging and laboratory testing in the USA is
often limited by insurance coverage, but in this study
was fully supported by the sponsor. Second, the
generalisability of the cancer detection rate is
constrained by the little ethnic, racial, and socioeconomic
diversity of the study cohort and the high rate of baseline
adherence to cancer screening. Similarly, volunteer bias
could have influenced participants’ risk or their
preferences for undergoing comprehensive diagnostic
assessments. The number, types, and stages of cancers
detected reflect results of an initial screen of undetected
cancers and might not be representative of the results of
long-term screening. Third, the study was done during
the COVID-19 pandemic, which hindered the ability to
obtain diagnostic assessments promptly and might have
lengthened the diagnostic resolution intervals. Fourth,
there was no structured evaluation system to elicit
www.thelancet.com Vol 402 October 7, 2023

adverse event reports, which might have led to
underestimation. Fifth, the study was not designed to
assess acceptability or cost-effectiveness. Finally, the
12-month follow-up period was short, and rates of later
cancer diagnoses are uncertain.
Although this study establishes clinical feasibility of
MCED testing, forthcoming results of larger studies
will be required to show clinical utility and effects on
cancer mortality. Three other studies are evaluating the
refined version of the MCED test developed in
PATHFINDER in different populations: (1) the
PATHFINDER2 cohort study (NCT05155605) is
investigating test performance in a larger and more
diverse US adult population, (2) the UK’s National
Health Service NHS Galleri study (ISRCTN 91431511) is
investigating test performance in 140 000 adults
randomly assigned to usual cancer screening versus
usual screening supplemented by annual MCED
testing,33 and (3) the University of Oxford’s SYMPLIFY
study (ISRCTN10226380) investigated the use of MCED
testing for individuals presenting to primary care
doctors with non-specific symptoms such as weight
loss or fatigue.34 Results of these studies will contribute
evidence regarding the utility of this MCED test in
specific clinical contexts.
In conclusion, PATHFINDER establishes the feasibility
of MCED testing in ambulatory practice and describes
the diagnostic assessments undertaken in response to
receipt of a positive test suggesting a possible cancer
diagnosis. This study also provides preliminary estimates
of test performance characteristics and the ultimate
diagnostic yield of this emerging technology. This
experience lays the foundation for larger-scale studies to
investigate the safety, utility, and clinical effectiveness of
MCED testing as a cancer screening strategy.
Contributors
EAK, CRM, KCC, ML, ETF, and DS conceptualised the study. ML and
ETF provided data curation. EAK, TMB, CHM, ML, ETF, and DS did the
formal analysis. EAK, TMB, CHM, LN, CAD, RR, CRM, and DS were
study investigators. EAK, TMB, ML, ETF, and DS contributed to
visualisation of the data and writing of the original draft. All authors
were involved in project administration, resourcing, and final review and
editing of the manuscript. EAK, KCC, ML, ETF, and DS accessed and
verified the data. All authors had access to the data, reviewed and
approved the final version of the manuscript, and were responsible for
the decision to submit for publication.
Declaration of interests
DS reports an uncompensated advisory role and serving as a study
investigator with research funding to Dana Farber Cancer Institute
(GRAIL) and personal fees for editorial service
(Journal of the American Medical Association). TMB reports a consultant
agreement with GRAIL, AbbVie, Amgen, Arvinas, Astellas Pharma,
AstraZeneca, Bayer, Bristol Myers Squib, Constellation, Dantari
Pharmaceuticals, GlaxoSmithKline, Janssen, Myovant Sciences, Pfizer,
Sanofi, and Sapience Therapeutics; and reports stock ownership in
Arvinas, Salarius Pharmaceuticals, and Exact Sciences. CHM reports a
consultant agreement with GRAIL. LN reports stock ownership in
Culmination Bio. KCC reports stock ownership in Illumina, Bristol
Myers Squib, Gilead, Baxter, and Bayer, and is an employee of GRAIL.
ETF and ML report stock ownership of Illumina and are employees of
GRAIL. EAK is an employee of GRAIL. All other authors declare no
competing interests.
www.thelancet.com Vol 402 October 7, 2023
Articles
Data sharing
The datasets analysed for this study are available upon reasonable
request by email to the corresponding author.
Acknowledgments
We acknowledge the contributions to this effort of the patients and their
families and the research coordinators and data managers at the
investigational sites. We would like to acknowledge Anne-Renee Hartman
for her contributions to the PATHFINDER study concept and design, and
Jafi Lipson for her contributions to the PATHFINDER study concept,
design, and implementation. We acknowledge Sana Raoof for her
contribution to the manuscript drafting. This study was sponsored by
GRAIL. We acknowledge Sarah A Prins (GRAIL) for manuscript editing
and management. We acknowledge Jennifer Hepker (Prescott Medical
Communications Group, Chicago, IL, USA) and Neva West (NeuroWest
Solutions, Seattle, WA, USA) for medical writing and editorial and
administrative support that was funded by GRAIL. DS was affiliated with
Dana Farber Cancer Institute and Harvard Medical School, Boston, MA,
USA, during the conduct of the study. TMB was affiliated with OHSU
Knight Cancer Institute, Portland, OR, USA, during the conduct of the
study. EAK was affiliated with Cleveland Clinic, Cleveland, OH, USA,
during the conduct of the study.
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