Pathology (February 2022) 54(1), pp.

6–19

GUIDELINES

BRAF mutation testing for patients diagnosed with stage

III or stage IV melanoma: practical guidance for the
Australian setting
RICHARD A. SCOLYER1,2,3,4,5, VICTORIA ATKINSON6,7,8, DAVID E. GYORKI9,10,
DUNCAN LAMBIE11,12, SANDRA O’TOOLE2,3,5, ROBYN P. M. SAW1,2,5,13,
BENHUR AMANUEL14,15, CHRISTOPHER M. ANGEL9, ALISON E. BUTTON-SLOAN16,
MATTEO S. CARLINO1,2,17,18, SYDNEY CH’NG1,2,5, ANDREW J. COLEBATCH3,5,
DARIUSH DANESHVAR2,19, INÊS PIRES DA SILVA1,2,17, TAMARA DAWSON20,
PETER M. FERGUSON1,2,3,5, ERWIN FOSTER-SMITH21, STEPHEN B. FOX9,22,
ANTHONY J. GILL2,3,23, RUTA GUPTA2,3,5, MICHAEL A. HENDERSON9,10,
ANGELA M. HONG1,2,13, JULIE R. HOWLE1,2,18, LOUISE A. JACKETT2,24,
CRAIG JAMES25, C. SOON LEE2,5,26,27,28,29, ALISTAIR LOCHHEAD30,31,32,
DAPHNE LOH33, GRANT A. MCARTHUR9,10, CATRIONA A. MCLEAN34,35,
ALEXANDER M. MENZIES1,2,13,36, OMGO E. NIEWEG1,2,5, BLAKE H. O’BRIEN37,
THOMAS E. PENNINGTON1,2,5, ALISON J. POTTER1,3,4,38, SAURABH PRAKASH39,
ROBERT V. RAWSON1,2,3,5, REBECCA L. READ40,41, MICHAEL A. RTSHILADZE1,5,13,
KERWIN F. SHANNON1,2,5, B. MARK SMITHERS7,8, ANDREW J. SPILLANE1,2,13,36,
JONATHAN R. STRETCH1,2,5, JOHN F. THOMPSON1,2,5, PAUL TUCKER42,
ALEXANDER H. R. VAREY1,2,18, RICARDO E. VILAIN3,43,44,
BENJAMIN A. WOOD15,45, GEORGINA V. LONG1,2,4,13,36
1
Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia; 2Faculty of
Medicine and Health, The University of Sydney, Sydney, NSW, Australia; 3NSW Health Pa-
thology, Sydney, NSW, Australia; 4Charles Perkins Centre, The University of Sydney, Sydney,
NSW, Australia; 5Royal Prince Alfred Hospital, Sydney, NSW, Australia; 6Princess Alexandra
Hospital & Greenslopes Private Hospital, Brisbane, Qld, Australia; 7Queensland Melanoma
Project, Princess Alexandra Hospital, Brisbane, Qld, Australia; 8Faculty of Medicine, Uni-
versity of Queensland, Brisbane, Qld, Australia; 9Peter MacCallum Cancer Centre,
Melbourne, Vic, Australia; 10Sir Peter MacCallum Department of Oncology, University of
Melbourne, Melbourne, Vic, Australia; 11Anatomical Pathology, Princess Alexandra Hospi-
tal, Pathology Queensland, Brisbane, Qld, Australia; 12University of Queensland Diamantina
Institute, Brisbane, Qld, Australia; 13Mater Hospital, North Sydney, NSW, Australia; 14School
of Medical and Health Sciences, Edith Cowan University, WA, Australia; 15PathWest Lab-
oratory Medicine WA, Nedlands, WA, Australia; 16Australia Melanoma Consumer Alliance,
Melbourne, Vic, Australia; 17Blacktown Hospital, Sydney, NSW, Australia; 18Westmead
Hospital, Sydney, NSW, Australia; 19Pathology West, Tissue Pathology & Diagnostic
Oncology, ICPMR-Westmead, Sydney, NSW, Australia; 20Melanoma & Skin Cancer Advo-
cacy Network, Melbourne, Vic, Australia; 21SA Pathology, Royal Adelaide Hospital,
Adelaide, SA, Australia; 22University of Melbourne, Melbourne, Vic, Australia; 23Kolling
Institute of Medical Research, Royal North Shore Hospital, Sydney, NSW, Australia; 24Austin
Pathology, Melbourne, Vic, Australia; 25Clinpath Pathology, Mile End, Adelaide, SA,
Australia; 26School of Medicine, Western Sydney University, Campbelltown, Sydney, NSW,
Australia; 27Ingham Institute for Applied Medical Research, Liverpool, Sydney, NSW,
Australia; 28Liverpool Hospital, Liverpool, Sydney, NSW, Australia; 29South Western Sydney
Clinical School, University of New South Wales, Sydney, NSW, Australia; 30Southern.IML
Pathology, Coniston, NSW, Australia; 31The Wollongong Hospital, Wollongong, NSW,
Australia; 32University of Wollongong, Wollongong, NSW, Australia; 33ACT Pathology,
Canberra Hospital, Canberra, ACT, Australia; 34Anatomical Pathology Department, Alfred
Hospital, Melbourne, Vic, Australia; 35Department of Medicine Central Clinical School,
Monash University, Melbourne, Vic, Australia; 36Royal North Shore Hospital, St Leonards,
Sydney, NSW, Australia; 37Sullivan Nicolaides Pathology, Brisbane, Qld, Australia;
38
Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia;
39
Melbourne Pathology (Sonic Healthcare), Melbourne, Vic, Australia; 40Calvary Health
Care, Canberra, ACT, Australia; 41College of Health and Medicine, Australian National
University, Canberra, ACT, Australia; 42Hobart Pathology, Hobart, Tas, Australia;

Print ISSN 0031-3025/Online ISSN 1465-3931 © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.
DOI: https://doi.org/10.1016/j.pathol.2021.11.002

43
School of Medicine and Public Health, University of Newcastle, Newcastle, NSW,
Australia; 44John Hunter Hospital, Newcastle, NSW, Australia; 45The University of Western
Australia, Perth, WA, Australia
Summary
Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now
among the possible treatment options for patients with
BRAF mutation-positive stage III or stage IV melanoma.
This makes prompt BRAF mutation testing an important
step in the management of patients diagnosed with stage III
or IV melanoma; one that can help better ensure that the
optimal choice of systemic treatment is initiated with minimal
delay. This article offers guidance about when and how
BRAF mutation testing should be conducted when patients
are diagnosed with melanoma in Australia. Notably, it rec-
ommends that pathologists reflexively order BRAF mutation
testing whenever a patient is found to have American Joint
Committee on Cancer (AJCC)/Union for International
Cancer Control (UICC) stage III or IV melanoma (i.e., any
metastatic spread beyond the primary tumour) and that
patient’s BRAF mutation status is hitherto unknown, even if
BRAF mutation testing has not been specifically requested
by the treating clinician (in Australia, Medicare-subsidised
BRAFV600 mutation testing does not need to be requested
by the treating clinician). When performed in centres with
appropriate expertise and experience, immunohistochem-
istry (IHC) using the anti-BRAF V600E monoclonal antibody
(VE1) can be a highly sensitive and specific means of
detecting BRAFV600E mutations, and may be used as a rapid
and relatively inexpensive initial screening test. However,
VE1 immunostaining can be technically challenging and
difficult to interpret, particularly in heavily pigmented tu-
mours; melanomas with weak, moderate or focal
BRAFV600E immunostaining should be regarded as equiv-
ocal. It must also be remembered that other activating
BRAFV600 mutations (including BRAFV600K), which account
for ~10–20% of BRAFV600 mutations, are not detected with
currently available IHC antibodies. For these reasons, if
available and practicable, we recommend that DNA-based
BRAF mutation testing always be performed, regardless of
whether IHC-based testing is also conducted. Advice about
tissue/specimen selection for BRAF mutation testing of pa-
tients diagnosed with stage III or IV melanoma is also offered
in this article; and potential pitfalls when interpreting BRAF
mutation tests are highlighted.
Key words: Stage IV melanoma; stage III melanoma; metastatic melanoma;
pathology; diagnosis; treatment; adjuvant; melanoma; BRAF mutation testing;
BRAF mutation-positive melanoma; targeted therapy.
Received 15 November, accepted 21 November 2021
Available online 20 December 2021
INTRODUCTION
Cutaneous melanoma is among the most aggressive forms of
skin cancer and its incidence is continuing to increase
BRAF MUTATION TESTING FOR STAGE III OR IV MELANOMA 7
worldwide.1–3 In Australia, a nation in which the incidence of
melanoma has historically been the highest, it is anticipated
that ~16,900 individuals will be diagnosed with melanoma in
2021 (approximately 5–10% with stage III or IV disease;
Fig. 1) and that >1300 may die from advanced-stage disease
during this same 12-month period.3,4 Therefore, melanoma
represents a significant and increasing public health
burden.1,2
Prior to 2010, systemic treatment options for patients with
American Joint Committee on Cancer/Union for International
Cancer Control stage III or IV disease remained limited to
chemotherapy, interleukin-2 and interferon alfa; progression-
free survival was often in the range of a few weeks to a few
months and the median overall survival (OS) for patients with
stage IV disease was typically less than 12 months.5,6 Since
that time, the development of new systemic drug treatment
options for metastatic melanoma, including immune check-
point inhibitors [e.g., anti-programmed cell death protein 1
(anti-PD-1) therapy, anti-cytotoxic T-lymphocyte antigen-4
(anti-CTLA-4) therapy] and targeted therapies (see below),
has dramatically improved outcomes in this clinical
setting.7–11 Indeed, when used to treat unresectable stage III
or stage IV melanoma, 2-year OS rates in excess of 50% have
been observed during phase III clinical trials of these more
recent treatment options, with a 5-year OS rate of 52% being
achieved in one trial of combination checkpoint inhibitor
therapy.7,12–15
Compared with other tumour types, cutaneous melanoma
is characterised by a high prevalence of somatic, tumouri-
genic mutations and the majority harbour mutations, such as
BRAF mutations, that affect the mitogen-activated protein
kinase (MAPK) signaling pathway (Fig. 2).7,16–22 BRAF
mutations result in constitutive activation of the BRAF pro-
tein in this pathway and downstream signalling of MEK and
ERK, promoting cellular proliferation, survival and migra-
tion.7,8,16–23 Approximately 35–50% of melanomas are
BRAF mutation-positive. In ~80–90% of cases, the BRAF
mutation is a BRAFV600E mutation; less commonly identified
variants include BRAFV600K [(~8–20%), BRAFV600R (1%),
BRAFV600M (<1%) and BRAFV600D (<1%) mutations, as well
as non-V600 BRAF variants (<1%).16–18,23–31
BRAF-mutated melanoma is often associated with
distinctive clinicopathological features.16,24–26,32–36 For
instance, compared with patients with BRAF wild-type mel-
anoma, those with BRAFV600E mutated disease tend to be
younger (50 years) and more likely to present with primary
tumours located on the trunk or other intermittently sun-
exposed anatomical sites.16,24–26,32–36 They are also more
likely to display certain histopathological features, including
fewer markers of chronic sun damage in the surrounding skin,
superficial spreading melanoma subtype, a high degree of
pagetoid spread, heavy melanin pigmentation, large epithe-
lioid cells, circumscription, prominent nesting, ulceration
and/or increased epidermal thickening.16,24–27,32–36 In
contrast, patients with BRAFV600K mutations often differ
8 SCOLYER et al. Pathology (2022), 54(1), February

Fig. 1 Differentiating between stage I/II, stage III and stage IV melanoma: Current American Joint Committee on Cancer staging criteria (8th edition).89,90 Adapted
from Gershenwald et al.90

from those with BRAFV600E mutation-positive melanomas; 53% relative reduction in risk of disease recurrence or death
e.g., they are more likely to be older (as are patients with (vs placebo) over a median 2.8-year follow up (primary
BRAFV600R mutations), have primary melanomas on the head analysis; Fig. 3) and a 49% relative reduction in risk of this
and neck region, and exhibit a higher degree of chronic sun same endpoint (vs placebo) at the time of subsequent 5-year
damage.25,34,37 follow-up analyses (hazard ratio = 0.51; 95% confidence
Several targeted therapy options aimed at inhibiting or interval 0.42–0.61].46,47
interrupting the mutationally-driven effects in BRAF-mutated
metastatic melanoma have been developed. These include a Current treatment recommendations for BRAF-mutant
range of selective BRAF kinase inhibitors (dabrafenib, disease
vemurafenib, encorafenib), as well as inhibitors of the
Clinical guidelines in Australia, Europe and the United States
downstream MEK kinase (trametinib, cobimetinib, binime-
now recommend the following as first-line systemic treat-
tinib). Multiple studies have shown that combination targeted
ment for patients with unresectable BRAFV600 mutation-
therapy with a BRAF inhibitor and a MEK inhibitor is more
positive stage III or IV melanoma: targeted therapy (BRAF
effective than use of a BRAF inhibitor alone and can also
inhibitor plus MEK inhibitor; or single-agent BRAF inhibitor
help reduce the frequency of particular side effects that are
if use of a MEK inhibitor is contraindicated); immunotherapy
sometimes associated with BRAF inhibitor monotherapy,
(anti-PD-1 receptor monotherapy ± anti-CTLA4 therapy); or
such as cutaneous squamous cell carcinoma.7,12,38–43
enrolment in a clinical trial.48–51 Targeted therapy is now
Importantly, targeted therapy is effective not only in pa-
considered a standard of care for patients with BRAF
tients with BRAFV600E mutant melanoma, but also in patients
mutation-positive advanced melanoma. Australian and in-
with the less common BRAFV600 mutation variants in
ternational guidelines also now indicate that either combined
advanced-stage melanoma, although it may be less effective
targeted therapy (BRAF inhibitor plus MEK inhibitor) or
than when used to treat BRAFV600E mutation-positive
immunotherapy (anti-PD-1 therapy) may be considered as an
melanoma.44,45
adjuvant treatment option for patients with BRAFV600
More recently, systemic targeted therapy (BRAF inhibitor
mutation-positive resected stage III disease.48,50,51
plus MEK inhibitor) has also been shown to significantly
In Australia, BRAF inhibitor/MEK inhibitor combination
improve patient outcomes when compared with placebo as
therapy is among a growing range of government-subsidised
adjuvant therapy for resected stage III melanoma (BRAFV600E
systemic treatments now available to eligible patients who
or BRAFV600K mutation-positive), being associated with a
have stage III or IV melanoma, via the national

Fig. 2 The mitogen-activated protein kinase (MAPK) signalling pathway.17 Note that in the presence of a BRAF mutation, inappropriate constitutive activation of the
pathway occurs and this leads to uncontrolled cell proliferation. RTK, receptor tyrosine kinase. Adapted from Zhou et al.17
Pharmaceutical Benefits Scheme (PBS).52 The most recent
development in this regard has been the inclusion of dabra-
fenib plus trametinib as a PBS-funded adjuvant targeted
therapy option for eligible patients with resected BRAFV600
mutation-positive stage IIIB, IIIC or IIID melanoma. It is now
not uncommon for patients with stage III melanoma to be
offered adjuvant therapy (targeted therapy or immuno-
therapy); at some institutions, all patients with stage III dis-
ease are encouraged to see a medical oncologist to discuss
adjuvant therapy options.
Practical implications of targeted therapy for BRAF-
mutant melanoma
Given the range of systemic treatment options that are now
potentially available for patients with stage III or IV mela-
noma, establishing the BRAF mutation status of a patient’s
disease has become an important step in the management of
metastatic melanoma.16,17,48–51 Knowing the BRAF mutation
status of newly diagnosed metastatic melanoma helps clini-
cians and patients make appropriately informed treatment
decisions and ensures that the optimal choice of systemic
treatment – if clinically indicated – can be initiated with
minimal delay.16,17,49–51
On their own, clinico-pathological features are neither
sensitive nor specific enough to reliably determine a mela-
noma patient’s BRAF mutation status. For this reason, we
BRAF MUTATION TESTING FOR STAGE III OR IV MELANOMA 9
strongly endorse current guidelines [e.g., European Society
for Medical Oncology (ESMO), National Comprehensive
Cancer Network (NCCN)],49,50 which recommend that BRAF
mutation testing be performed routinely whenever patients
have stage III or IV melanoma and initiation of targeted
systemic therapy for BRAF mutation-positive disease may be
an option or the testing may help confirm eligibility for
enrolment in a clinical trial.49,50 To facilitate such testing, in
Australia, patients diagnosed with stage III or IV melanoma
(i.e., any patient whose melanoma has spread beyond the
primary site) are now eligible for Medicare-subsidised
BRAFV600 mutation testing. Importantly, such testing does
not need to be requested by the treating clinician; it may also
be independently ordered by a pathologist.53
Unfortunately, despite its importance, available data/
anecdotal reports suggest that, in Australia, newly diagnosed
stage III or IV melanomas are not always routinely sent for
BRAF mutation testing (BRAF mutation status was estab-
lished by pathologists in 87% of cases in one study54) and/or
that BRAF mutation test results are not always available when
initial decisions regarding the management of a patient’s
stage III or IV melanoma are being made. This article offers
guidance about when and how BRAF mutation testing should
be conducted for melanoma patients in Australia, with the
aim of improving rates of BRAF mutation testing when it is
indicated and better ensuring that the BRAF mutation status
10 SCOLYER et al. Pathology (2022), 54(1), February

Fig. 3 Adjuvant BRAF inhibitor plus MEK inhibitor therapy for patients with stage III BRAF mutation-positive melanoma: impact on relapse-free and overall sur-
vival.46 Data are from the COMBI-AD study, a double-blind, placebo-controlled, phase III clinical trial, in which patients with completely resected, stage III melanoma
with BRAFV600E or BRAFV600K mutations were randomised to receive either oral dabrafenib (150 mg twice daily) plus trametinib (2 mg once daily) or two matched
placebo tablets for 12 months. Relapse-free survival = primary endpoint; overall survival (OS) = secondary endpoint. Figure shows Kaplan–Meier estimates of relapse-
free survival and OS (intention-to-treat population). Median follow up = 2.8 years; median OS had not been reached in either group. Adapted from Long et al.46

Fig. 4 Guide to BRAF mutation testing for patients diagnosed with melanoma. AJCC, American Joint Committee on Cancer.
of individual patients’ stage III or IV melanoma is known
when initial treatment decisions are being made.
WHEN TO CONDUCT BRAF MUTATION
TESTING
BRAF mutation status is currently the only validated pre-
dictive biomarker of melanoma response to BRAF inhibitor/
MEK inhibitor therapy, and any delay in confirming it could
potentially delay the initiation of the optimal treatment
(which could adversely affect achieved clinical outcomes,
BRAF MUTATION TESTING FOR STAGE III OR IV MELANOMA 11
especially for patients with advanced-stage or rapidly pro-
gressive disease).7,16,17 For this reason, whenever a patient is
diagnosed with stage III or IV melanoma (i.e., any metastatic
spread beyond the primary tumour), rapid determination of
BRAF mutation status (i.e., at the time of diagnosis) should be
considered a priority (Fig. 4).48–51,55
Clinicians (e.g., surgeons, medical oncologists, dermatol-
ogists, general practitioners) should routinely indicate to
pathologists that BRAF mutation testing needs to be con-
ducted if a patient is found to have stage III or IV melanoma
12 SCOLYER et al.
and that patient’s BRAF mutation status is hitherto unknown.
In addition, if a treating clinician suspects that the patient may
have a melanoma metastasis (and that patient’s BRAF mu-
tation status is hitherto unknown), the pathology request for
the corresponding biopsy should state this and ask that BRAF
mutation testing be performed. Although structured pathol-
ogy reporting protocols published by the Royal College of
Pathologists of Australasia56 (Supplementary Fig. 1,
Appendix A) include ‘BRAF mutation status’ as a
recommended/non-core element that needs to be routinely
checked and reported when a pathology specimen contains
metastatic melanoma, it is the view of the authors that this
should be a required/core element of the pathology report for
patients with stage III or IV melanoma.
Reflex testing at time of diagnosis of metastatic
melanoma is recommended
We strongly recommend that pathologists reflexively order
BRAF mutation testing whenever a patient is found to have
stage III or IV melanoma (i.e., any metastatic spread beyond
the primary tumour) and that patient’s BRAF mutation status is
hitherto unknown (e.g., if the result of any earlier BRAF mu-
tation test result does not appear on the request form or in
laboratory records), even if BRAF mutation testing has not
been specifically requested. If the BRAF mutation status of a
patient’s melanoma has been determined previously, then it is
suggested that the result of this earlier testing be clearly indi-
cated on the pathology request form; doing this will alert the
pathologist not to proceed with reflex BRAF mutation testing.
It can perhaps be reasonably assumed for the great majority of
patients undergoing a sentinel node biopsy that BRAF muta-
tion testing is unlikely to have been previously performed (as
such testing is not recommended for patients with stage I or II
disease); if sentinel node positivity is confirmed in this situa-
tion, then the pathologist would be the first to know that the
patient has stage III disease and well-placed to be able to
judiciously initiate BRAF mutation testing earlier than any
other member of that patient’s care team.
Importantly, our recommendation that pathologists
reflexively order BRAF mutation testing when patients are
found to have stage III melanoma extends to patients who
have stage IIIA disease (including all with a single positive
sentinel lymph node and those with an in-transit, satellite or
Fig. 5 Examples of positive BRAF mutation test results using immunohistochemistry (IHC). (A) Metastatic melanoma within a lymph node (stage III) showing strong
and diffuse positive IHC staining with the anti-BRAF V600E (VE1) antibody, indicating the presence of a BRAFV600E mutation. (B) Metastatic melanoma showing very
weak positive staining with the anti-BRAF V600E (VE1) antibody; such staining should be interpreted as equivocal and BRAF mutation status should be determined by a
DNA-based testing method.
Pathology (2022), 54(1), February
microsatellite metastasis in isolation; Fig. 4). In Australia,
adjuvant therapy is not currently government-subsidised for
patients with BRAF mutation-positive stage IIIA melanoma.
However, at least some of these individuals may still seek
systemic treatment and be willing to self-fund it. Individual
patients with confirmed BRAF mutation-positive stage IIIA
disease may also be eligible for a clinical trial.
One of the benefits of reflex BRAF mutation testing, in
addition to ensuring that a patient’s BRAF mutation status is
established as soon as possible, is that it potentially obviates
the need to conduct BRAF mutation testing at a later time
(which may involve having to retrieve archived tissue and/or
present logistical challenges). Knowledge of a patient’s BRAF
mutation status is also likely to help inform discussions about
that particular patient during multidisciplinary team meetings.
If/when BRAF mutation testing is initiated by a patholo-
gist, it is recommended that both the testing modality and the
way the result will be reported (e.g., via a supplementary
histopathology report or a separate molecular pathology
report) be documented in the histopathology report.
Proposed reporting timeframe
For patients diagnosed with stage IV melanoma, the results of
BRAF mutation testing may be critical for determining the next
treatment step and required urgently in some cases. With this
in mind, we recommend that pathology laboratories seek to
ensure a turnaround time of 10 consecutive calendar days
from the time that a specimen is received. We also recommend
that, when a test result is required urgently, clinicians indicate
this on the pathology request form and/or contact the reporting
pathologist directly to stress the urgent clinical need and/or
discuss what might be done to help expedite the testing.
The need for a quick turnaround may not be as great when
a patient has stage III versus stage IV melanoma. For cases
where the need for a result is less urgent, we suggest that an
acceptable turnaround time is 14 consecutive calendar days
from the time a specimen is received by the laboratory; once
again, if more rapid turnaround is required, then this should
be clearly communicated by the clinician.
Sourcing or retrieving specimens from other pathology
laboratories can be challenging and presents one of the
biggest potential barriers to rapid BRAF mutation testing.
Requests to retrieve or send melanoma specimens from one
 BRAF MUTATION TESTING FOR STAGE III OR IV MELANOMA 13

Fig. 6 Integrative Genomics Viewer image of two metastatic melanomas harbouring a BRAFV600E mutation (c.1799T>A, chr7:140,453,136, GRCh37/hg19), detected
using a next-generation sequencing panel (VariantPlex, ArcherDx). (A) A melanoma case with a mutant allele frequency of 66% and (B) a melanoma case with a mutant
allele frequency of 16%. Image courtesy of Dr James Wilmott, Melanoma Institute Australia.

laboratory to another should always be treated as ‘urgent
requests’ and expedited accordingly, as patient management
may be impacted by delays. Laboratories that host the spec-
imen being sought should make an effort to expedite the
retrieval, shipment or postage of that specimen if/when it is
requested.
BRAF MUTATION TESTING IN PRACTICE
Selecting a BRAF mutation testing technique
There are many BRAF mutation testing techniques, but the
two broad options include: (a) protein-based testing; or (b)
DNA-based testing.16–18 Each of the available options has
different technical and practical advantages, as well as certain
limitations.
BRAF mutation testing should always be conducted by an
accredited laboratory (e.g., in Australia, a facility accredited
by National Association of Testing Authorities), using an
approved and appropriately validated test, which is also
subject to ongoing quality assurance checks and audits. The
optimal choice of BRAF mutation test depends on multiple
factors, such as its sensitivity (its ability to identify BRAF
mutations with a low rate of false negatives), specificity (its
ability to identify BRAF mutations with a low rate of false
positives), limit of detection (the threshold at which the signal
defining a positive or negative test can be distinguished from
the background) and anticipated turnaround time. An addi-
tional consideration is the ability of the testing modality to
detect BRAFV600 mutations other than the BRAFV600E
variant, remembering that ~10–20% of BRAFV600 mutations
are BRAFV600nonE variants (e.g., BRAFV600K).16–18,23–31
Protein-based testing
The only protein-based test currently available for BRAF
mutation testing is an immunohistochemistry (IHC)-based
test that involves the use of an anti-BRAF V600E
14 SCOLYER et al.
monoclonal antibody (VE1) to stain BRAFV600E-mutant
protein (Fig. 5).16–18,55,57–60 It has the advantage of being a
relatively simple, broadly accessible and inexpensive test,
and typically ensures a rapid turnaround time.51 Importantly,
it is also both highly sensitive (97–100%) and highly specific
(92–100%) for detecting BRAFV600E mutations.16,57–65
Available data indicate that its sensitivity can be at least
comparable with that of DNA-based testing modalities, while
its specificity may sometimes be slightly lower. In one study,
researchers found that IHC could detect BRAFV600E muta-
tions with 100% sensitivity and 100% specificity when they
defined a positive test as strong immunostaining (3+) in
80% of tumour cells.55,66 For these reasons, provided the
testing laboratory is appropriately accredited and implements
robust quality assurance measures to ensure the testing is
reliable (and remains so over time), IHC-based testing may be
used as a rapid and relatively inexpensive initial screening
test for patients who have stage III or IV melanoma and may
be potentially eligible for targeted therapy. For BRAFV600E
IHC to be interpreted as positive, it must be strongly (3+) and
diffusely positive (Fig. 5A). In all other instances of positive
staining, including where there is weaker or patchy positivity
(Fig. 5B), the test should be interpreted as equivocal.58,60,67,68
The use of illustrative unequivocally positive (and
sequencing confirmed) external controls may aid in deter-
mining the staining threshold required for a BRAFV600E IHC
result to be interpreted as positive. An important potential
pitfall when interpreting BRAFV600E IHC is the presence of
melanin pigmentation within melanoma tumour cells, which
may result in a false positive result if it is not recognised.
A notable drawback of the currently available IHC-based
BRAF mutation test is that it can only detect BRAFV600E
mutations and not other BRAFV600 mutations that may still
respond to targeted therapy. IHC-based testing may also
return false positive or false negative results in a small pro-
portion of cases (e.g., false negative results might occur if the
staining area is necrotic/pre-necrotic; a false positive result
may occur if it is heavily melanin-pigmented, depending on
the staining technique or where weak or heterogeneous
staining is interpreted as positive).18,50,55,59,60 For this reason,
if available and practicable, and even if an IHC-based test
results in strong and diffuse positive staining (and abundant
melanoma pigment is not present), we recommend that
confirmatory testing using a DNA-based technique always be
ordered, provided a sufficient quantity of suitable tissue is left
for such analyses (i.e., to corroborate the IHC-based finding
and also check for other BRAF mutations).16–18,50,57,60
Indeed, many centres in Australia routinely conduct DNA-
based testing regardless of whether IHC-based testing is
also performed.
DNA-based molecular testing
DNA-based testing techniques are advanced testing modal-
ities that identify the order of specific nucleotides in tumour
cell DNA and can reveal the presence (or absence) of
particular genetic mutations (Fig. 6).16,55,69 The more
commonly utilised DNA-based modalities include real-time
polymerase chain reaction testing, next-generation
sequencing, Sanger sequencing, mass spectroscopy and
pyrosequencing.
DNA-based testing is considered the ‘gold standard’
testing modality for the detection of BRAF mutations,16,55,69
Pathology (2022), 54(1), February
but it often takes more time than IHC-based testing (delaying
turnaround time) and is not always readily accessible/
routinely available in all pathology laboratories. In addition,
some diagnostic samples may be unsuitable for DNA-based
testing because they contain insufficient tumour cells
(which can lead to false negative test results)58,70 or, in some
instances, because they have undergone processing that has
compromised the integrity of the DNA in the sample (e.g.,
biopsies taken from bone tissue may need to be de-calcified
prior to testing, which can cause DNA fragmentation; the
decalcification process may also result in false negatives
when conducting IHC-based testing).71
In the event that both DNA-based and IHC-based BRAF
mutation testing is performed (e.g., if confirmatory DNA-
based testing is performed following an initial IHC-based
test), it is recommended that pathologists and clinicians
seek to confirm whether the results are concordant, consider
possible reasons for any discordance (if discordance is
identified) and correctly document the overall BRAF muta-
tion status for the patient. Doing this will help further mini-
mise the risk of a treatment decision being made on the basis
of a false positive or false negative test finding.
Selecting the specimen
BRAF mutation testing is usually carried out on formalin-
fixed and paraffin-embedded melanoma specimens, but can
also be performed on fresh or fresh-frozen tissue. Any
specimen, including a cytological specimen (obtained via fine
needle aspiration) or core biopsy sample, is potentially suit-
able for BRAF mutation testing, provided it contains suffi-
cient tumour cells, but histological specimens are
preferred.18,50
Depending on the testing modality that is being used, there
will be specific requirements regarding the quantity and
quality of tissue that should be used (typically specified in the
validated protocol for each test). In general, the more tumour
cells that are present in a specimen (i.e., the greater the
neoplastic cellularity), the more reliable a BRAF mutation test
result is likely to be. For this reason, particularly when using
DNA-based modalities, we recommend against testing any
specimen for which the tumour cell component represents
less than 20% of all cells in the specimen (unless a more
suitable specimen is not available, in which case the labora-
tory conducting the testing would need to be aware of the low
tumour cell content and select an appropriately sensitive
testing modality). Care should also be taken to avoid testing
necrotic tissue, fatty tissue, haemorrhagic tissue or melanin-
rich areas (as the presence of heavy pigmentation may
affect the accuracy of DNA-based tests and the interpretation
of IHC findings55,57). For these and other reasons, selection
of the specimen to be tested should always be based on
careful morphological assessment. Macrodissection may
need to be considered in some cases, to enhance tumour
purity.
For patients with metastatic melanoma, it is recommended
that the sample to be tested is the most readily available
current stage III or IV disease specimen.18,50 If such a sample
is not available or not suitable for testing (e.g., due to
insufficient neoplastic cellularity), the testing may be
performed on a sample obtained from a previous biopsy or
even from the primary melanoma tumour specimen.18 This is
because the BRAF mutation status of a melanoma is almost

always retained across all stages of disease.18,59,67,68,70,72–76
It has been noted that many studies reporting discordance of
BRAF mutation status between the primary and metastatic
melanoma have been conducted using less sensitive DNA-
based testing methods, which can yield false negative re-
sults when samples contain insufficient tumour content. In
contrast, those revealing a high level of concordance have
tended to be those involving the use of IHC-based testing, the
accuracy of which is less reliant on there being a certain
percentage of tumour cells present.67,70 Intra-patient discor-
dance between the primary and metastatic melanoma speci-
mens may also occur if the primary tumour being tested is not
the tumour from which the metastatic disease was derived
(~10–12% of melanoma patients develop multiple primary
tumours).70,77,78 Thus, it is highly probable that the BRAF
mutation status of a patient’s primary and metastatic tumours
will be the same, provided the specimens being tested contain
an adequate quantity of viable tumour cells, there is minimal
admixture of non-tumour cells and the primary tumour being
tested is the tumour from which the metastasis was
derived.67,70
It is important to note that small metastases in sentinel
lymph node (SLN) biopsies are often unsuitable for mutation
testing, particularly when using DNA-based modalities,
because small numbers of tumour cells may be admixed with
numerous non-tumour cells and this may result in a false
negative result. For this reason, testing of small SLN me-
tastases should be avoided, if possible (i.e., unless no alter-
native is available or unless a bulky sentinel node metastasis
is present); it is better to test the primary tumour specimen in
this situation, if available. In such instances, IHC-based
testing may be particularly useful for identifying
BRAFV600E mutations (if present) because the BRAFV600E
status of small sentinel lymph node metastases can be accu-
rately determined with careful morphological evaluation by
an experienced pathologist.
If a specimen is unsuitable for testing (e.g., due to the very
small size of the metastasis) and no other sample is imme-
diately available, it is recommended that the pathologist
specifically note this in the pathology report, thus alerting the
treating clinician to decide whether there is a need to initiate
separate testing of another specimen (e.g., the primary
tumour, if practicable). For patients with a bulky stage III or
stage IV melanoma, BRAF mutation status may also be
determined by analysis of circulating tumour DNA (ctDNA)
in a peripheral blood sample.79–81 Analysis of ctDNA is
currently not routinely performed but it may have a role in
future clinical practice, particularly when an appropriate
tissue biopsy of the melanoma is not readily available for
mutation testing.
Other tests that may be requested when patients have
melanoma
In Australia, BRAF mutation status is currently the only
validated, ‘actionable’ biomarker that can be confirmed using
a Medicare-reimbursed test and used to help identify those
patients with metastatic melanoma who are likely to benefit
from BRAF inhibitor/MEK inhibitor therapy. No other mu-
tations/biomarkers currently have utility in the metastatic
melanoma setting as a means of identifying patients who
might benefit from an Australian Therapeutic Goods
Administration (TGA)-approved, PBS-subsidised treatment.
BRAF MUTATION TESTING FOR STAGE III OR IV MELANOMA 15
For this reason, BRAF mutation testing remains the only
mutation test that we recommend be routinely, reflexively
ordered when patients are diagnosed with stage III or IV
melanoma.
Nevertheless, we do suggest that clinicians always
consider requesting additional testing if/when it is possible to
do so and the results have the potential to influence treatment
recommendations (e.g., if they suggest there may be benefit
in trying a particular non-PBS-subsidised treatment that the
patient is willing to self-fund, etc.). Current guidelines
(ESMO, NCCN) recommend that broader genomic profiling
be considered if/when the results might guide future treat-
ment decisions or confirm eligibility for participation in a
clinical trial.49,50 Conducting such additional testing may also
assist in the collection of data that helps identify potential
future therapeutic targets.
In terms of which specific additional tests could or should
be conducted, potentially relevant genes of interest might
include c-KIT and NRAS, as well as NF1, GNAQ and/or
Box 1. Key points and recommendations
 Combination targeted therapy (BRAF inhibitor plus MEK inhibi-
tor) is now among the possible treatment options for patients with
BRAF mutation-positive stage III or IV melanoma and the avail-
ability of this treatment means BRAF mutation testing is now an
essential step in the management of patients diagnosed with meta-
static melanoma; accurate confirmation of BRAF mutation status is
crucial for optimal personalisation of melanoma therapy.
 Prompt initiation of BRAF mutation testing when patients are
diagnosed with stage III or stage IV melanoma (i.e., any metastatic
spread beyond the primary melanoma) better ensures that the optimal
choice of systemic treatment can be initiated with minimal delay.
 In Australia, patients diagnosed with stage III or IV melanoma (i.e.,
any metastatic spread beyond the primary melanoma) are now
eligible for Medicare-subsidised BRAF V600 mutation testing;
importantly, this testing may be independently ordered by a
pathologist.
 We strongly recommend that pathologists reflexively order BRAF
mutation testing when a patient is found to have stage III or IV
melanoma (i.e., any metastatic spread beyond the primary mela-
noma) and that patient’s BRAF mutation status is hitherto unknown
(even if BRAF mutation testing has not been specifically requested by
the treating clinician).
 For patients with stage III/IV melanoma (i.e., any metastatic spread
beyond the primary melanoma), it is recommended that BRAF mu-
tation testing be conducted on a sample obtained via biopsy of the
most readily available current stage III or IV disease; if such a sample
is not available or if it is deemed unsuitable for testing (e.g., due to
insufficient neoplastic cellularity), the testing may be performed on a
sample obtained from a previous biopsy or even from the primary
tumour.
 Immunohistochemistry (IHC) can play a useful role in the
assessment of patients’ BRAF mutation status (e.g., as an initial
screening test, when DNA-based testing is not readily available or
practicable); however, we recommend that DNA-based testing
always be ordered if/when practicable, regardless of whether IHC-
based testing is also conducted, to corroborate any initial IHC-based
finding and/or check for BRAF mutations not detectable via IHC
(e.g., BRAFV600K).
16 SCOLYER et al.
GNA11. For example, the detection of c-KIT mutations may
guide the selection/use of a tyrosine kinase inhibitor; it has
been suggested that NRAS mutations, which are frequently
detected in melanoma (~15–20% of cases), may increase
sensitivity to MEK inhibitor therapy, and clinical trials of
targeted therapy in patients with NRAS-mutant melanoma are
currently underway; also, uveal melanomas may harbour
GNAQ or GNA11 mutations, which can lead to constitutive
activation of the MAPK pathway.16,28,49,82–88
In the future, testing for activating gene fusions, such as
those involving NTRK, ALK, BRAF and ROS1, and tumour
mutation burden may have therapeutic relevance.
BRAF mutation testing for earlier-stage (primary)
melanoma
It has been suggested that BRAF mutation testing should
always be performed at the time of primary melanoma diag-
nosis because knowledge of it will be useful if the patient
subsequently develops metastatic disease. However, the great
majority of patients are diagnosed with a primary melanoma at
an early clinical stage and will never develop metastatic dis-
ease. In addition, there is currently no available evidence that
use of systemic BRAF inhibitor therapy, based on the results
of a BRAF mutation test, offers clinical benefit in the early-
stage (I/II) melanoma setting (i.e., when there is no evidence
of metastatic spread beyond the primary tumour). Therefore, it
is not currently recommended that BRAF mutation testing be
routinely ordered on every primary melanoma (stage I/II
melanoma patients) and, in fact, such testing methods would
also not currently be Medicare-subsidised (publicly funded) in
Australia in this setting. Once again, this is consistent with
clinical practice guidelines (e.g., NCCN guidelines), which
recommend that primary melanomas be tested for BRAF mu-
tations only when such testing is required to guide the choice
of systemic therapy and/or when checking a patient’s eligi-
bility for enrolment in a clinical trial.50
Clinical trials of systemic therapy, including BRAF in-
hibitors and MEK inhibitors, are now underway in patients
who have been diagnosed with stage II melanoma. These and/
or future trials may result in growing availability and/or
approval of targeted therapy options for patients in this
earlier-stage disease setting. As a result, advice about whether
and when patients’ primary melanoma should be routinely
sent for BRAF mutation testing may need to be appropriately
revised in the future.
CONCLUSION
In an era when combination targeted therapy (BRAF inhibitor
plus MEK inhibitor) is now among the possible treatment
options for patients with BRAF mutation-positive stage III or
stage IV melanoma, timely and accurate confirmation of
BRAF mutation status is crucial for optimal personalisation of
their melanoma therapy. For this reason, BRAF mutation
testing is now a recommended—and essential—step in the
management of patients diagnosed with stage III or IV mel-
anoma (Box 1).
In Australia, Medicare-subsidised BRAF mutation testing
of stage III or IV melanoma specimens may now be inde-
pendently ordered by a pathologist and, for this reason, we
strongly recommend that pathologists reflexively order BRAF
mutation testing whenever a patient is found to have stage III
or IV melanoma and that patient’s BRAF mutation status is
Pathology (2022), 54(1), February
hitherto unknown. BRAF mutation testing should preferably
be conducted on a sample of the most readily available cur-
rent stage III or IV specimen (if available and suitable for
testing) and, if necessary (e.g., if DNA-based testing is not
readily available or practicable), may be performed using
IHC in the first instance, with the results subsequently
confirmed using a DNA-based technique. Following these
recommendations will help better ensure that the optimal
choice of systemic treatment is initiated with minimal delay
and improve melanoma patient management.
Acknowledgements: The image in Fig. 6 was kindly
provided by Dr James Wilmott, Melanoma Institute
Australia. The authors thank Tim Brereton for writing
assistance. They also are grateful for support from
colleagues at their respective institutions.
Conflicts of interest and sources of funding: In May 2021,
Novartis Pharmaceuticals Australia convened and funded a
meeting of specialists with multidisciplinary expertise to
discuss BRAF mutation testing in Australia. The meeting was
chaired by RAS and VA, DEG, DL, SO, RPMS and GVL
participated and contributed to the discussions. This manu-
script was developed following the meeting and is reflective
of the discussions that occurred at it. Subsequently, all co-
authors reviewed and provided feedback on the manuscript.
Novartis manufactures dabrafenib and trametinib which
are licensed and reimbursed in Australia for the treatment of
eligible patients with BRAF V600-positive unresectable/
metastatic melanoma (stage III/IV) and for the adjuvant
treatment of eligible patients with involvement of the lymph
node(s), following complete resection. While Novartis
supported the production of this manuscript, neither the
company nor any of its representatives had any input into its
content beyond the provision of requested factual
information.
RAS has received fees for professional services from
Evaxion, Provectus Biopharmaceuticals Australia, QBiotics,
Novartis, Merck Sharp & Dohme, NeraCare, Amgen Inc.,
Bristol-Myers Squibb, Myriad Genetics and GlaxoSmithK-
line. DEG has received honoraria for advisory board partic-
ipation from Amgen, QBiotics and Bayer, and speaking
honoraria from Bristol-Myers Squibb and Merck Sharp &
Dohme. SOT has received honoraria from Roche, Bristol-
Myers Squibb and Novartis. RPMS has received honoraria
for advisory board participation from Merck Sharpe &
Dohme, Novartis and QBiotics Group Limited, and speaking
honoraria from Bristol-Myers Squibb and Novartis. BA has
received research funding from AstraZeneca, Merck Sharpe
& Dohme and Roche; and has been on advisory boards for
AstraZeneca, Merck Sharpe & Dohme and Roche. MSC has
been an advisory board member for Amgen, Bristol-Myers
Squibb, Eisai, Ideaya Biosciences, Merck Sharpe &
Dohme, Nektar, Novartis, OncoSec, Pierre Fabre, QBiotics,
Regeneron, Roche, Merck and Sanofi, and has received
honoraria from Bristol-Myers Squibb, Merck Sharpe &
Dohme, and Novartis. IPD has received travel support from
Bristol-Myers Squibb and Merck Sharp & Dohme, and
speaker fees from Roche, Bristol-Myers Squibb, Merck
Sharp & Dohme and Novartis. SF has received honoraria for
advisory board participation and/or research funds (to insti-
tution) from Novartis, Roche, Bayer, Bristol-Myers Squibb,

Merck Sharpe & Dohme, AstraZeneca, Amgen, Glax-
oSmithKline, Janssen and Thermo Fisher Scientific. AMH
has received honoraria for advisory board participation from
QBiotics Group Limited and Oncobeta. GAM is a principal
investigator for clinical trials with Array Biopharma/Pfizer
and Roche/Genentech. All revenues were paid to his insti-
tution as reimbursement for trial costs. He has been a non-
reimbursed advisor for Novartis and Bristol Myers Squibb.
AMM has received honoraria for advisory board participation
from Bristol-Myers Squibb, Merck Sharpe & Dohme,
Novartis, Roche, Pierre-Fabre and QBiotics. RLR has
received speaking honoraria from Bristol-Myers Squibb.
KFS has received honoraria for advisory board participation
and speaking honoraria from Merck Serono. AJS has
received an honorariam from Eli Lilly. JFT has received
honoraria for Advisory Board participation from Merck
Sharpe & Dohme Australia and Bristol Myers Squibb
Australia, honoraria and travel expenses from Glax-
oSmithKline and Provectus Inc, and support for conference
attendance from Novartis. GVL is consultant advisor for
Aduro Biotech Inc, Amgen Inc, Array Biopharma inc,
Boehringer Ingelheim International GmbH, Bristol-Myers
Squibb, Evaxion Biotech A/S, Hexel AG, Highlight Thera-
peutics SL, Merck Sharpe & Dohme, Novartis Pharma AG,
OncoSec, Pierre Fabre, QBiotics Group Limited, Regeneron
Pharmaceuticals Inc, SkylineDX BV, and Specialised Ther-
apeutics Australia Pty Ltd.
RAS is supported by an NHMRC Practitioner Fellowship
(APP1141295) and is a recipient of an NHMRC Program
Grant (APP 1093017). RPMS is supported by Melanoma
Institute Australia. GAM is supported by the Lorenzo and
Pamela Galli Medical Research Trust. AMM is supported by
Nicholas and Helen Moore, and Melanoma Institute
Australia. AJP is funded by Deborah McMurtrie and John
McMurtrie AM, through Melanoma Institute Australia. RVR
is supported by a Clinical Research Scholarship from Sydney
Research. JRS is supported by Melanoma Institute Australia.
GVL is supported by an NHMRC Practitioner Fellowship
and the University of Sydney Medical Foundation and is a
recipient of an NHMRC Program Grant (APP 1093017). JFT
is a recipient of an NHMRC Program Grant (APP 1093017).
Tim Brereton (who was funded by Novartis Pharmaceuticals
Australia) provided writing assistance.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.pathol.2021.11.002.
Address for correspondence: Prof Richard A. Scolyer, Royal Prince Alfred
Hospital, Missenden Road, Camperdown, NSW, 2050, Australia. E-mail:
richard.scolyer@health.nsw.gov.au
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