10 1111@his 13248

DR NAVEENA SINGH (Orcid ID : 0000-0003-1782-1967)
DR GLENN MCCLUGGAGE (Orcid ID : 0000-0001-9178-4370)

Accepted Article
Article type : Review
TITLE PAGE
TITLE: HIGH GRADE SEROUS CARCINOMA (HGSC) of TUBO-OVARIAN

ORIGIN: RECENT DEVELOPMENTS
AUTHORS: Naveena Singh1, W Glenn McCluggage2, C Blake Gilks3
1
Department of Cellular Pathology, Barts Health NHS Trust, London, UK 2 Department of
Pathology, Belfast Health and Social Care Trust, Belfast, UK, 3 Department of Anatomic
Pathology, 1st Floor JPPN, Vancouver General Hospital, Vancouver, Canada
Corresponding author:
Naveena Singh
Department of Cellular Pathology
Barts Health NHS Trust
2nd Floor, 80 Newark Street
London E1 2ES
UK
Tel: (44) 2032460217/0198

Fax: (44) 2032460202
Email: N.Singh@bartshealth.nhs.uk
Key words: High grade serous carcinoma, HGSC, pathology, immunohistochemistry,

diagnosis, genetic abnormalities
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/his.13248
This article is protected by copyright. All rights reserved.
ABSTRACT
Extra-uterine high-grade serous carcinoma (HGSC) accounts for most of the morbidity and
mortality associated with ovarian carcinoma, and is one of the leading causes of cancer death
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in women. Until recently our understanding of HGSC was very limited, compared to other
common cancers, and it has only been in the last 15 years that we have learned how to
accurately diagnose this ovarian carcinoma histotype. Since then, however, there has been
rapid progress, with identification of a precursor lesion in the fallopian tube, development of
prevention strategies for both those with inherited susceptibility (Hereditary Breast and
Ovarian Cancer Syndrome) and without the syndrome, and elucidation of the molecular
events important in oncogenesis. This molecular understanding has led to new treatment
strategies for HGSC, with the promise of more to come in the near future. In this review we
focus on these recent changes, including diagnostic criteria/differential diagnosis, primary
site assignment, precursor lesions, and the molecular pathology of HGSC.
INTRODUCTION
A major triumph of recent decades has been our increasing knowledge of the molecular
events underlying cancer. In the field of ovarian cancer clinico-pathological observations,
including the identification of “early” cancers in risk reducing salpingo-oophorectomy
(RRSO) specimens in patients with BRCA1/2 germline mutation, coupled with molecular
studies have transformed our understanding of the origins and biology of epithelial ovarian
cancers (EOC). Long considered a single disease with protean morphology, EOC is now
separated into five distinct major histological types (1). The differing biology of these
histological types has direct implications for their prevention, treatment and genetic risk
assessment. Accurate diagnosis underpins their correct management in current practice. The
distinction between these cancer types and their clinical outcomes are blurred in historic data,
and much of the morbidity and mortality of EOC is now realised to be due to a single entity,
high-grade serous carcinoma (HGSC). This review highlights recent developments
concerning the diagnosis, origin, classification and molecular pathology of HGSC. Going
forward it is critical for histopathologists to not only diagnose HGSC and other EOCs
accurately, but also to understand their underlying molecular events, as the frontier shifts
towards molecular subclassification of HGSC in order to provide enhanced prognostic and
predictive information that will better guide patient management.

CURRENT CLASSIFICATION OF EPITHELIAL OVARIAN CANCERS
The 2014 World Health Organisation (WHO) Classification includes five major categories of
EOC, namely high grade serous, low grade serous, endometrioid, clear cell and mucinous
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carcinomas (2). Their diagnosis has management implications as these tumours differ with
respect to their sensitivity to classical platinum-based chemotherapy, radiotherapy and
targeted treatments. In addition their diagnosis signifies differences in underlying risk for
hereditary cancer syndromes; while a diagnosis of HGSC should mandate risk assessment for
BRCA1/2 or related abnormalities, a diagnosis of non-HGSC, principally endometrioid and
clear cell carcinomas, should result in assessment for Lynch Syndrome (3).
The 2014 Classification reflects several important changes in our understanding of EOC. In
the prior 2003 Classification, there was only a single category of serous carcinoma (4) and
the division into low grade serous carcinoma (LGSC) and HGSC is a culmination of the now
firmly established fact that these represent two distinct tumour types.
Previously it was considered that the various subtypes of EOC were part of a spectrum of
disease with the same origin, postulated to be the ovarian surface epithelium, that had the
potential for multiple lines of differentiation resulting in the common occurrence of mixed
EOCs. Indeed, up until 15 or 20 years ago, mixed EOCs, such as mixed serous and
endometrioid or mixed serous and clear cell, were diagnosed not uncommonly. However,
recent studies using modern diagnostic criteria, supplemented by immunohistochemistry and
molecular interrogation, have shown that mixed EOCs are very uncommon, accounting for
<1% of EOCs (5, 6).
A relatively recent population based study which included central pathology review using
modern diagnostic criteria provides updated information regarding the relative frequencies of
the major subtypes of EOC (7) (table 1) with HGSC comprising 68.1% of EOCs and LGSC
3.4% (4). This study showed an increase and decrease in the frequencies of serous and
endometrioid carcinomas respectively; this reflects the previous poorly reproducible
distinction between HGSC and high grade endometrioid carcinoma (EC) (8-12) and the
realisation that many neoplasms previously diagnosed as high grade EC were, in fact,
HGSCs. Similarly it is recognised that most tumours previously diagnosed as transitional cell
carcinoma are in fact HGSC. This study also showed that 88.7% of advanced stage EOCs
were HGSC (7).

Thus HGSC constitutes by far the commonest of 5 distinct EOC types and accounts for most
of the morbidity and mortality associated with carcinoma of tubo-ovarian origin.
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CONCEPT OF LOW GRADE AND HIGH GRADE SEROUS CARCINOMA
The perceived relationship between benign, borderline and malignant ovarian serous
neoplasms was controversial for many years and it was always tempting to speculate that a
continuum existed from benign to borderline to malignant. However, pathological evidence
for this was lacking and it was generally assumed that there was no relationship between
borderline and malignant serous neoplasms, although occasionally the two were found to
coexist. Recent studies have convincingly demonstrated that there are two distinct types of
ovarian serous carcinoma, LGSC and HGSC (13-23). Although termed low grade and high
grade, it is stressed that these are not two grades of the same neoplasm but rather two
different tumour types, both with a distinct underlying histogenesis and associated with
different molecular events during oncogenesis, behaviour and prognosis. HGSC is much
more common than LGSC by a factor of around 20 to 1 (68.1% HGSC and 3.4% LGSC in
the population based study referred to earlier (7)). LGSC arises in many cases in a stepwise
fashion from a benign serous cystadenoma through a serous borderline tumour to an invasive
low grade serous carcinoma with a well-defined adenoma-carcinoma sequence. However, it
is not proven that all LGSCs arise from a pre-existing serous borderline tumour (SBT), with
SBT identified in approximately 80% of cases of LGSC (24), and it is possible that some do
not. In contrast, HGSC is not related to serous borderline tumour and there is now
overwhelming evidence (discussed later) that most HGSCs originate from the epithelium of
the distal fimbrial portion of the fallopian tube. The classification of a serous carcinoma as
low grade or high grade has been shown to be reproducible amongst pathologists (25, 26),
although there are occasional cases where this distinction may be problematic (discussed
below). Almost all serous carcinomas which would have previously been classified as
moderately or poorly differentiated represent HGSCs while those which would have been
classified as well differentiated/grade 1 may be either LGSC or HGSC using the two tier
classification. For example, some architecturally well differentiated serous carcinomas have
high nuclear grade and represent examples of HGSC.

MORPHOLOGICAL FEATURES OF HGSC
HGSC is characterised by morphological heterogeneity, both within and between individual
neoplasms. There is often an admixture of different architectural patterns including papillary,
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micropapillary, slit-like, glandular, microcystic/ microglandular, solid and transitional-like
(Figure 1). While papillary elements and slit-like spaces are characteristic of HGSC, well-
formed rounded glands are also common, resulting in a pseudoendometrioid appearance. The
tumour cells usually have relatively scant cytoplasm but occasionally there is abundant
eosinophilic or clear cytoplasm, or cells with vacuoles and intracytoplasmic mucin, resulting
in a signet ring appearance. The neoplastic cells consistently show high-grade nuclear
features, with significant pleomorphism and a high mitotic rate with frequent abnormal
mitotic figures. As a rule, high power microscopic fields with multiple mitoses are easily
found. Often individual bizarre tumour giant cells are present. Areas of necrosis and
psammoma bodies are common (but the presence of numerous psammoma bodies should
raise the possibility of LGSC). These architectural and cytological patterns frequently coexist
in a tumour. This variability in tumour appearance (intra-tumoral heterogeneity) is one of the
characteristic features of HGSC and can result in morphological confusion with other
subtypes of EOC as discussed later. In particular it is important to recognise that the
combination of ‘Solid, pseudo-Endometrioid and Transitional-like’architectures, abbreviated
as ‘SET’ morphology, together with strikingly high mitotic activity, frequent bizarre mitoses
and abundant tumour-infiltrating lymphocytes, is seen typically, but not exclusively, in cases
with germline or somatic BRCA-related abnormalities (27). Over 90% of tubal/ ovarian
HGSCs are WT1 positive, usually, but not always, diffusely so. They almost all exhibit
“mutation-type” p53 staining (given the ubiquitous presence of TP53 mutations), although
there are occasional exceptions (discussed later). p16 exhibits diffuse “block-type”
immunoreactivity in about two-thirds of HGSCs while most of the remainder exhibit focal
immunoreactivity (28, 29).
PROBLEMATIC AREAS IN HISTOLOGICAL TYPING OF EOCs

Previously there was considerable interobserver variability amongst pathologists in the typing
of EOCs (8-12). The situation has improved remarkably and recent studies have shown that
with modern diagnostic criteria, excellent interobserver agreement can be achieved amongst
specialist gynaecological and general pathologists in the typing of EOCs (30-32). Much of
the improved interobserver agreement relates to the fact that it is now realised that most high
grade advanced stage EOCs are HGSCs, and WT1 immunohistochemical staining has been

instrumental in establishing this as it is a reliable, although not totally specific, marker of
serous carcinomas, both LGSC and HGSC (33-37). In the next sections, the distinction
between HGSC and the other major subtypes of EOC is covered, with the exception of
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mucinous carcinoma that does not typically enter into the differential diagnosis of HGSC.
Table 2 lists markers that may assist in the various diagnostic scenarios.
Distinction between HGSC and LGSC

In most cases, it is straightforward to distinguish between HGSC and LGSC on
morphological examination alone. However, there are occasional cases where this may be
problematic, especially on small biopsy specimens such as a core biopsy (38). Difficulties
may arise because some HGSCs have deceptively bland monomorphic cytology with quite
regular appearing nuclei and relatively low mitotic activity. This may result in close
morphological mimicry of LGSC, especially when there is prominent micropapillary
architecture comprising micropapillary structures surrounded by clefts, a growth pattern
characteristic of LGSC (Figure 2a). While in a substantial tissue specimen, there are usually
areas of more overt nuclear atypia with tumour giant cells, significant mitotic activity and
necrosis, these features may not be present in a limited biopsy specimen. Conversely, in some
LGSCs the nuclei are larger than is usual with prominent nucleoli and appreciable mitotic
activity (Figure 2b); in such cases, there is a risk of overdiagnosing HGSC.
In such problematic cases, the only useful marker in distinguishing LGSC from HGSC is
p53, with “wild-type” staining in the former and “mutation-type” in the
latter. It is stressed that it is no longer appropriate to report p53
staining as positive or negative. Instead, p53 staining is referred to as
abnormal, aberrant or “mutation-type” (a pattern of staining which is
highly predictive of underlying TP53 mutation) as opposed to normal or
“wild-type” (a pattern of staining predictive of intact or wild-type TP53
gene) (39). There are two major patterns of “mutation-type” p53 staining:
(i) diffuse intense immunoreactivity involving greater than 80% of tumour
cell nuclei and (ii) totally negative (“null” pattern) staining of tumour
cell nuclei associated with a positive internal control in the form of weak
staining of a proportion of non-neoplastic cells, for example stromal or
lymphoid cells; the two patterns of “mutation-type” staining are referred
to as “all or nothing staining” (40). In contrast, a “wild-type” pattern
comprises heterogeneous nuclear staining, ranging from a few positive cells
to almost all cells showing some staining but of variable intensity, and

with less than 80% of nuclei showing strong staining. It should be noted
that 5% of HGSC show “wild-type” p53 immunostaining in the presence of an
underlying TP53 mutation (39); thus while “mutation-type” staining pattern
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strongly supports a diagnosis of HGSC rather than LGSC, “wild-type” p53
immunostaining is not totally specific in the distinction between LGSC and
HGSC, as it can be seen in either. An additional, but rare, abnormal,
aberrant or “mutation-type” staining pattern is cytoplasmic with variable
nuclear expression (39). These abnormal patterns reflect the nature of the
underlying TP53 mutation (table 3).
Transformation of LGSC into High Grade Carcinoma

Although HGSC and LGSC represent two distinct tumour types with a different underlying
pathogenesis, there are rare cases where a LGSC or SBT coexists with or transforms into a
high-grade carcinoma, that can show morphological features of HGSC,
undifferentiated/anaplastic carcinoma or carcinosarcoma (41-43). In such cases, it is likely
that the high-grade carcinoma arises from the low-grade serous neoplasm. There is a risk of
overdiagnosing a component of high-grade carcinoma in a LGSC since, as discussed, in the
latter there may be foci exhibiting nuclear enlargement with some degree of mitotic activity.
It is emphasised that high-grade transformation of LGSC is rare and should be diagnosed
with caution. Unfortunately, p53 immunohistochemistry does not help in most of these cases
in that true high-grade transformation of a low-grade serous neoplasm is often not associated
with TP53 mutation and thus p53 staining is often “wild-type” (41-43). The molecular
mechanisms underlying the high-grade transformation in such cases is currently unknown;
however, the absence of TP53 abnormalities indicate that they are distinct from usual HGSC,
where TP53 mutations are ubiquitous.
Distinction between HGSC and Endometrioid Carcinoma

Previously some pathologists tended to diagnose many high grade, non-mucinous ovarian
carcinomas as serous in type while others classified them as endometrioid or mixed serous
and endometrioid. The prevailing view, supported by convincing evidence, is that the vast
majority represent HGSCs. In this distinction, WT1 immunohistochemical staining may be
of value (30-33, 35). Over 90% of primary ovarian/ tubal HGSCs exhibit nuclear positivity
with WT1 that is usually, but not always, diffuse, while most endometrioid carcinomas (EC)
are negative or at the most focally positive (33, 35, 37). In problematic cases, WT1 staining
is recommended as an adjunct to help distinguish between HGSC and high grade EC. Of

note, some low grade ECs are WT1 positive, meaning that this marker is of less value in the
distinction between a LGSC and a low grade EC (44). As discussed, almost all HGSCs
exhibit “mutation-type” p53 staining. However, high grade ECs, which uncommonly occur as
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primary ovarian neoplasms, may exhibit either “mutation-type” or “wild-type”
immunoreactivity. Other morphological pointers such as squamous differentiation, an
adenofibromatous pattern and background endometriosis/endometriotic cyst, should also be
taken into account and, if present, may assist in making a diagnosis of primary ovarian EC.
Distinction between HGSC and Clear Cell Carcinoma

Ovarian clear cell carcinoma (CCC) characteristically shows an admixture of growth patterns
with tubulocystic, papillary and solid architectures and eosinophilic stromal hyalinisation.
CCCs are usually diagnosed at low stage (I or II), in contrast to HGSC (7). Most CCCs are
diagnosed without difficulty but previously there was a tendency to overdiagnose CCC or a
mixed HGSC and CCC due to the presence of clear cells within HGSCs (6). The presence of
areas of more typical HGSC is a useful feature and it is stressed that the mere presence of
clear cells does not constitute CCC. WT1 and ER are usually negative in CCCs, p16 is
usually negative or focally positive and p53 exhibits “wild-type” staining; as discussed, most
HGSCs are diffusely positive with WT1 and p16 and there is “mutation-type” staining with
p53. Napsin A, hepatocyte nuclear factor 1 beta and racemase are useful markers of ovarian
CCC, especially the first of these (45-49). It should be noted that a subset of CCC show
abnormal “mutation-type” p53 staining (46) despite other morphological and
immunohistochemical features favouring CCC; in tumours of the endometrium such cases
have been shown to demonstrate more aggressive clinical behaviour and this should be stated
in the report (50).
Transitional Cell Carcinoma Is Removed From The WHO 2014 Classification Of

Ovarian Carcinomas
Transitional cell carcinoma (TCC) is not included in the 2014 WHO Classification of EOCs
(2), having been part of the 2003 Classification (4). This is because almost all tumours that
were previously diagnosed as TCC represent variants of HGSC, or less commonly ECs, with
a transitional-like architecture (51-53). So-called TCCs of the ovary are usually positive with
WT1, (52, 54), and some recur or metastasise as typical HGSC, both factors favouring their
being variants of HGSC. The WHO 2014 classification includes the category of malignant

Brenner tumour, which arises from a benign and/ or borderline counterpart and for which
morphological criteria have been clearly defined (2).
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Undifferentiated Carcinoma
The WHO definition of an undifferentiated ovarian carcinoma is a malignant epithelial
tumour showing no differentiation of any specific Mullerian cell type (2). Most probably
represent the extreme end of the spectrum of HGSC in which solid elements are often present
and almost always when these neoplasms are sampled well areas diagnostic of HGSC, such
as vague papillary formations, slit-like spaces or psammoma bodies, will be identified. A
morphologically undifferentiated ovarian carcinoma which is diffusely positive with WT1
and exhibits “mutation-type” p53 staining can be reasonably reported as solid pattern HGSC.
A smaller number of undifferentiated ovarian carcinomas represent dedifferentiation within a
low grade EC (the combination of low grade endometrioid and undifferentiated carcinoma is
referred to as dedifferentiated carcinoma) and the low grade component can be totally
overgrown; the concept of dedifferentiation within uterine and ovarian ECs, especially the
former, has been highlighted in recent years (55). Most examples of this type of
undifferentiated carcinoma (and the undifferentiated component of dedifferentiated
carcinomas), in contrast to solid pattern HGSC, are WT1 negative and do not show
“mutation-type”-type p53 immunostaining/TP53 mutation. Instead it has been recently
demonstrated that, in common with ovarian small cell carcinomas of hypercalcemic type,
these often exhibit loss of expression of encoding proteins in the SWI/SNF chromatin
modelling complex (SMARCA4, SMARCB1) and/or DNA mismatch repair gene proteins
(56, 57).
MIXED EPITHELIAL OVARIAN CARCINOMAS ARE EXTREMELY

UNCOMMON
It is now recognized that mixed EOCs are rare and, in fact, this designation has been dropped
from the 2014 WHO Classification (2). In older studies there was a relatively high frequency
of mixed EOCs (3-11%) (5) and it was not uncommon for pathologists to make a diagnosis of
a mixed serous-endometrioid carcinoma, a mixed serous-clear cell carcinoma or a mixed
serous-transitional carcinoma. The diagnosis of mixed EOC historically was based on routine
histological examination. However, it is apparent that each of the major subtypes of EOC can
have a broad spectrum of morphological appearances that can result in close mimicry of other

subtypes. The demonstration of immunohistochemical differences between the different
morphological subtypes has resulted in refinement of diagnostic criteria with the result that
mixed EOCs are now considered to be very uncommon. EOCs with admixed areas of classic
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HGSC, “endometrioid-like” areas, “clear cell” areas and “transitional-like” areas are now
appreciated to almost always represent HGSCs rather than mixed EOCs. A recent study of
871 EOCs, combining modern diagnostic criteria and detailed molecular interrogation using
immunohistochemistry, gene expression and hotspot sequence analysis, concluded that the
incidence of true mixed carcinomas is <1% (5), and most represent combinations of
carcinomas that occur in endometriosis. In this study the combination of EC and CCC was
found to be the most common mixed EOC.
OVERWHELMING EVIDENCE FOR FALLOPIAN TUBE ORIGIN OF

EXTRAUTERINE HGSC
There is now irrefutable evidence that most extrauterine HGSCs arise from the fallopian tube,
most commonly the fimbrial end, from a precursor lesion referred to as serous tubal
intraepithelial carcinoma (STIC) (58-63). Although there is still variation in the awareness
and acceptance of the evidence for a tubal origin, this continues to change as new evidence
emerges. A recent survey amongst members of national and international gynaecological
pathology and clinical societies indicated widespread acceptance among both pathologists
and clinicians of the fallopian tube theory of origin of HGSC (86% pathologists, 92%
clinicians) (64). The evidence for tubal origin is discussed together with a proposal for
primary site assignment in extraovarian HGSC.
Traditional Theories of Site of Origin of “Ovarian HGSC”

Two related theories have dominated our thinking over the past decades (65). The first is that
all ovarian carcinomas arise from the ovarian surface epithelium (OSE), the mesothelium
covering the ovary and resting on specialized gonadal stroma. It was believed that in adult
life the OSE retained the embryological potential for multilineage differentiation and could
therefore give rise to a range of tumours of various Müllerian types, including mixed EOCs.
While this is an attractive theory as it explains the occurrence of a variety of Müllerian
tumours within an organ that does not normally contain Müllerian derivatives, evidence in
the form of identifying precursor lesions or early neoplasms is lacking. The second theory, an
expansion of the first, is that the totipotential property of OSE also extends to the

mesothelium lining the peritoneal cavity. This forms the basis of the “secondary Müllerian
system”. This theory provides a potential explanation for cases of tumour widely
disseminated within the peritoneum in the presence of no or minimal ovarian involvement
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and also the occurrence of HGSC many years after removal of the tubes and ovaries.
Fallopian Tube as Site of Origin of HGSC

It is almost universally accepted that the fimbrial end of the fallopian tube, is the site of
origin of a large majority of HGSCs in patients with a hereditary predisposition as evidenced
by the almost invariable tubal location of early HGSCs identified in risk-reducing salpingo-
oophorectomy (RRSO) specimens (58, 65-67). In order to extend this theory of tubal origin
to HGSCs occurring in women without a familial predisposition it needs to be determined:
(a) whether STIC and small tubal mucosal HGSCs are found in sporadic “ovarian” HGSCs at
comparable frequency to that in high-risk patients,
(b) whether the tubal lesions are clonally related to the tumour elsewhere or if they could
arise independently as part of a ‘field change’, and
(c) whether the tubal lesions precede the disease in the ovaries and other extratubal sites or if
these could be metastatic.
Each of these questions is considered below.
(a) Frequency of STIC and Tubal Mucosal Invasive Carcinomas in Sporadic/Non-High

Risk HGSC
The frequency of finding STIC and/or a small tubal mucosal invasive HGSC is dependent on
two important factors: how meticulously the fallopian tube is examined and the extent of
disease in tubes and ovaries. These are microscopic lesions and identification depends on
careful examination of the tubes by a method that maximizes the histological assessment of
the epithelium, particularly that covering the tubal fimbriae. The widely accepted SEE-FIM
(Sectioning and Extensively Examining the FIMbria) protocol (58) is recommended and
results in the finding of STIC or invasive tubal mucosal HGSC in up to 60% of cases of
sporadic advanced HGSC (60). In our experience, it is relatively unusual to find STIC alone
in cases of advanced HGSC; it is more common to identify a small HGSC involving the tubal
mucosa. In a prospective study of 53 chemonaive cases of extrauterine HGSC with full tubal
sampling using a SEE-FIM protocol (68), STIC alone was present in 9% (5/53) and tubal
invasive mucosal HGSC with or without STIC in 49% (26/53). In 25% of cases, the fallopian
tube (usually the fimbrial end) was partly or wholly incorporated into a mass, precluding

assessment of mucosal changes. The more advanced the growth of the primary tumour, the
less likely will be the detection of early tubal precursors as these are rapidly overgrown. Only
17% of cases showed no tubal involvement in the form of STIC, invasive mucosal HGSC or
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overgrowth of the tube by the tumour mass.
The opportunity to examine and characterise very early examples of sporadic HGSC
analogous to that seen in RRSO specimens arises occasionally when these are discovered
incidentally in a surgical specimen. Four recent studies have reported cases of STIC and/or
invasive mucosal HGSC diagnosed as incidental findings at surgery carried out for unrelated
reasons. These occurred in women who were not at high risk for ovarian cancer (69-72). The
tubal mucosa was involved in 100% (48 of 48) of cases and STIC, with or without an
invasive mucosal carcinoma, was identified in 98% (47 of 48).
These studies demonstrate a comparable incidence of STIC and tubal mucosal invasive
carcinoma in sporadic cases of HGSC, as compared with HGSCs in patients with BRCA1/2
germline mutations. They also provide compelling evidence that the earliest lesions in
sporadic HGSC are in the fallopian tube.
(b) Clonal Relationship Between STIC and Concurrent HGSC

HGSC exhibits a high degree of genomic diversity and this heterogeneity makes assessment
of a clonal relationship between STIC and concurrent HGSC complex (73, 74). Despite this
complexity, monoclonality of tubal mucosal, ovarian and extra-adnexal lesions has been
demonstrated both at presentation and recurrence (75). About 97% of extrauterine HGSCs
exhibit TP53 mutation as an early founder event and this may be the only somatic mutation
that is consistently present in all samples from a given case (73). Mutational analysis of
pelvic HGSC with concurrent STIC has shown these to harbour identical TP53 mutations in
the vast majority of cases (59, 76, 77). The demonstration of an identical TP53 mutation at
different sites is strong evidence of clonal identity, as the probability of an identical mutation
occurring simultaneously in multiple sites is extremely low. It can be summarised that there
is irrefutable evidence that HGSC arises from a single tumour clone, and that multiple foci of
disease within the peritoneal cavity do not result from multifocal origin or a “field change”.
(c) Evidence that STIC Represents the Primary Lesion

The spread of invasive carcinomas to distant sites where they demonstrate an intraepithelial
growth pattern and mimic a primary lesion at the metastatic site is a well-recognized
phenomenon. A recent report of 100 non-gynaecological tumours metastatic to the fallopian
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tubes showed 49% of the metastases to be fimbrial in location and 29% to be
mucosal/epithelial, often closely mimicking STIC (78). This raises the possibility that some
STIC lesions could be metastatic, and instances of STIC-like lesions metastatic from uterine
serous carcinoma are well described (79, 80).
It is challenging to demonstrate whether or not STIC represents a primary or metastatic lesion

since molecular analysis requires substantial well preserved cells, and these lesions are
minute and only detected on histological examination of formalin-fixed and paraffin
embedded tissue. Nevertheless this has been investigated with a variety of techniques. A
study comparing telomeric length in cases of STIC with concurrent HGSC found that STIC
lesions had the shorter telomeres, in keeping with a precursor-carcinoma relationship (81). A
study on multiple samples from the same patients confirmed the high level of intratumoural
genomic diversity in HGSC, but also that this diversity arises in early stages of disease
development (75). This study mapped the clonal ancestry of each lesion and the tubal
mucosal lesion was shown phylogenetically to be the earliest of multiple sites of disease
examined. Further evidence has been provided in a recent study on the gene encoding cyclin
E, CCNE1, and centrosome amplification in STIC and HGSC. CCNE1 copy number changes
were highly concordant in cases with concurrent STIC and HGSC. Gain or amplification of
CCNE1, occurred in 8/37 (22%) cases of STIC and 12/43 (28%) cases of HGSC, and
centrosome amplification was recorded in 5/37 (14%) and 10/25 (40%) of STIC and HGSC
cases respectively. The findings of this study suggest that CCNE1 copy number
gain/amplification is an early event in tumour progression and precedes centrosome
amplification. The finding of a lower prevalence of centrosome amplification in STIC than in
HGSC supports the view that STIC represents a precursor of HGSC (82).
Further evidence that STIC represents the primary lesion is provided by simpler
observational studies. It is recognised that bilaterality in ovarian carcinoma supports
secondary involvement; for example, metastatic colorectal, gastric, pancreatico-biliary, breast
and cervical cancers are often bilateral. However, pathologists have always ignored this for
HGSC. The incidence of bilateral involvement of the ovaries in HGSC is significantly higher

than in the tubes. In one study, 62% of ovarian HGSCs were bilateral while tubal
involvement was unilateral in 84% of cases (68). This difference in incidence of bilaterality
also favours a tubal origin with the ovarian involvement being secondary. Another
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observation favouring tubal origin comes from the reports of incidental HGSC cases (69-72).
Of the 48 cases included in these four papers, all 43 cases with unifocal, ie organ-confined,
disease showed fallopian tube mucosal involvement as the sole site of disease. There was
ovarian involvement in only 8% (4/48) of cases, always in association with tubal mucosal
involvement, and therefore likely to be secondary.
It is well recognized that the ovary frequently harbours metastases that far exceed the size of
the primary tumour, as exemplified by tumours of gastric, appendiceal, colorectal, cervical
and other origins. The ovaries are recognized to be a fertile soil for the growth of metastatic
epithelial cells and it has been postulated that this occurs due to the presence of enzymatically
active steroid-producing stromal cells (83, 84). It is interesting that while this phenomenon is
accepted without question for tumours metastasizing to the ovary from distant sites,
traditional dogma continues to cast doubt on this mechanism for HGSC, despite the lack of
evidence for ovarian origin in the majority of cases.
DIAGNOSIS OF STIC
Crucial to the identification of precursor lesions of HGSC in the fallopian tube is the correct
diagnosis of STIC. STIC comprises a population of cytologically malignant epithelial cells
replacing the normal tubal epithelium. It most commonly, but not exclusively, involves the
fimbriae, and is characterized by increased nuclear to cytoplasmic ratio with rounded nuclei,
loss of cell polarity, coarsely clumped chromatin, prominent nucleoli and the presence of
mitotic activity involving non-ciliated, ie secretory, cells. Other features that may be present
include epithelial stratification, small fracture lines in the epithelium and tufting and
exfoliation of small epithelial cell clusters from the epithelial surface. Earlier studies showed
significant interobserver variability in the diagnosis of STIC on haematoxylin and eosin
stained sections, even amongst specialist gynaecological pathologists (85). The diagnostic
criteria for STIC have since evolved and guidelines for diagnosis have been published, which
include the use of p53 and Ki67 immunostaining (85-87). Adherence to these criteria results
in a high degree of interobserver diagnostic agreement. In discrete fallopian tube mucosal
lesions with high-grade atypia, the presence of mutation-type p53 staining and a high Ki67
labelling index (> 10%) support a diagnosis of STIC (Figure 3). Although immunostains are

a valuable adjunct in the diagnosis of isolated lesions of the fallopian tube, they are usually
not needed to diagnose STIC in the context of advanced stage HGSC where comparison
between the tubal mucosal lesion and HGSC elsewhere reveals identical cytological features.
Accepted Article
In practice, it is also our view that immunohistochemistry is not necessary in cases where the
diagnosis of STIC is clear-cut. Immunohistochemistry can be reserved for those cases where
morphology alone is insufficient for unequivocal diagnosis and in such cases use of the
published imunostaining protocol is valuable (86, 87).
Fallopian tube epithelial lesions with atypia that do not meet all the criteria for STIC are
variously referred to as tubal intraepithelial lesion in transition (TILT) or serous tubal
intraepithelial lesion (STIL) (88-90). These lesions are of uncertain significance and, in our
opinion, these diagnostic terms should not be used in routine practice; additional research is
required to determine the clinical significance, if any, of such lesions. Similarly p53
signatures should not be reported. These take the form of small foci of intense p53 nuclear
staining involving consecutive secretory cells, most commonly but not exclusively in the
fimbriae, in the absence of cytological atypia (91). TP53 mutations have been demonstrated
in some p53 signatures and these may represent the earliest stage of development of HGSC.
However, p53 signatures are extremely common in the fallopian tube even in patients with
benign disease and no hereditary predisposition to developing HGSC and it is clear that only
a small proportion will ever develop into a STIC or HGSC (91).
Pathologists should also appreciate that the normal fallopian tube epithelium can exhibit a
degree of nuclear variation and atypia, especially in premenopausal patients. This can result
in overinterpretation and overdiagnosis of possible precursor lesions, especially since
pathologists are now scrutinizing the fallopian tube epithelium in a manner which was not
done previously. Benign alterations, such as cautery artefact, can also be misinterpreted as
precursor lesions. In such problematic cases, p53 and Ki67 immunohistochemistry is
recommended if there is doubt as to the diagnosis. p53 is especially useful since true
precursor lesions exhibit one of the patterns of “mutation-type” staining while benign lesions
exhibit “wild-type” immunoreactivity.
Another consideration is that fallopian tube mucosal involvement by uterine or non-
gynaecological primary tumours can occur and mimic STIC (78, 79). Most cases with
unilateral or bilateral HGSC in the ovary and/or STIC or HGSC in the tube but with an
endometrial serous intraepithelial or invasive carcinoma will represent adnexal metastases

from an endometrial primary (80). WT1 may be of value in these cases, although there is
significant overlap. Most tubal HGSCs exhibit diffuse nuclear positivity with WT1 while
most uterine serous carcinomas are negative. However, there is some overlap in that a
Accepted Article
proportion of uterine serous carcinomas are WT1 positive (the percentage has varied between
studies but may be up to 30%) and a small percentage of tubal HGSCs are WT1 negative (33,
92).
FALLOPIAN TUBE SAMPLING - WHAT IS THE OPTIMAL PROTOCOL?

As they are not visible grossly, an essential step in identifying STIC and small tubal mucosal
HGSCs is detailed examination of the tubes, especially the fimbriae, in cases of extrauterine
HGSC. The fimbriae were not routinely sampled for histological examination prior to the
realization that in RRSO specimens, this is the area most likely to harbour an abnormality. As
a result, more detailed sampling of the tube has been widely adopted using a protocol that
maximizes microscopic examination of the fimbrial epithelium (Sectioning and Extensively
Examining the FIMbriated end of the fallopian tube, SEE-FIM) (58). This requires
longitudinal sectioning of the distal fallopian tube at 2 mm intervals. It has also been
demonstrated that deep levels through the fimbrial blocks, in addition to standard single
sections, improves detection of STIC lesions and helps in identification of an associated
invasive component (93). In many cases of HGSC, there is complete or partial obliteration of
the tube within an obvious tubo-ovarian mass, making detailed sampling unnecessary. In
those cases where the tubes are clearly separable from the ovaries and macroscopically
normal, complete fimbrial sampling similar to that suggested in the SEE-FIM protocol is
recommended as a minimum, with examination of the rest of the tube if needed. Complete
examination of the fimbrial end of the tubes, with 3 additional deep levels, and examining the
rest of the tube if no lesion is detected will maximize detection of tubal involvement. We
acknowledge that such sampling will not change patient management in most cases, although
with disease confined to the tube, detecting a small tubal mucosal HGSC when only STIC
was present in original sections may do so since this will generally result in a
recommendation for chemotherapy. The method of sampling the tube significantly affects
site assignment in extrauterine HGSC (94).
There is also variability in the sampling of tubes removed at surgery for benign conditions,
including sterilization. With increased acceptance of the tubal origin of HGSC, opportunistic

salpingectomy is becoming increasingly common. While a SEE-FIM protocol has been
recommended (72), there is no evidence that this is cost-effective. This results in multiple
additional blocks per case, while the likelihood of detecting an incidental STIC or HGSC is
Accepted Article
very small. In a published study of 522 cases of women at low risk of ovarian cancer in
whom the entire tube was examined, STIC alone was detected in 3 cases (0.6%) (72), while a
prospective departmental study over a period of one year detected no lesions in 632 cases
with no known risk factors for ovarian cancer (CB Gilks, unpublished data). A study of 2268
cases over 6 years in a single institution demonstrated a 0.17% incidence of STIC in low risk
women (95), ie an occurrence of one in 590 cases. Furthermore, as there is no evidence that
adjuvant treatment is needed for patients with STIC only (96, 97), the benefits of detection
are negligible, at least until such time as the diagnosis has treatment implications. The
diagnosis of invasive HGSC is another matter, as these patients will typically be offered
adjuvant chemotherapy since, at present, it is not possible to identify a subset of patients with
invasive HGSC with a sufficiently low risk of recurrence that chemotherapy can be withheld.
Thus careful gross examination of the tubes at the time of surgical cut-up is important, with
sampling of any abnormalities. If there are no gross abnormalities, a single representative
block from the fimbriae should be sampled as that is where significant pathology occurs;
often this can encompass an intact or bisected entire fimbrial end. There is no rationale for
submitting a single routine section from the mid-portion of the fallopian tube, as was our
practice in the past, although many laboratories will still submit 1 or 2 transverse sections
from the non-fimbrial portion as well as the fimbriae.
PROPOSAL FOR UNIFORM SITE ASSIGNMENT IN EXTRAUTERINE HGSC

The updated FIGO 2014 system has unified the staging of primary ovarian, tubal and
peritoneal cancers (98). This is accompanied by the mandate that primary site should be
assigned as tubal, ovarian, peritoneal or undesignated but no guidance is offered for
assignment. In the 2014 WHO Classification, it is stated that site assignment of extraovarian
HGSC is left to the “experience and professional judgment” of the reporting
pathologist/tumour board (2). It is perhaps not widely known that the 2014 WHO “blue
book” has amended the criteria for making a diagnosis of primary peritoneal carcinoma
(PPC). While the Gynecologic Oncology Group (GOG) criteria were previously widely used
(defined by size criteria) (4, 99), PPC is currently defined by WHO as peritoneal HGSC when

both tubes and ovaries are grossly and microscopically normal (or enlarged by benign
disease) without tumour involvement (1).
Accepted Article
In the absence of an agreed protocol, there can be significant variability in the assignment of
site of origin of extrauterine HGSC by individual pathologists/tumour boards, with potential
for identical cases to be classified as primary ovarian, tubal, peritoneal or undesignated
(Figure 4). In a recent international survey amongst 173 pathologists and 101 clinicians, a
case with STIC and HGSC in the ovary was assigned as a tubal primary by 56% of
respondents and as an ovarian primary by 44%. Furthermore the same case scenario was
assigned as stage I by 45% of respondents, presumably those who assigned these as ovarian
primaries and do not consider STIC in the absence of invasive foci in the fallopian tube
sufficient to upstage the carcinoma, and stage II by 55%, since classifying such a case as
tubal carcinoma with ovarian spread necessarily means stage II disease (64).
The compelling evidence that the fallopian tube is the site of origin of most extrauterine
HGSCs should be acknowledged and reflected in our clinical practice by adhering to an
agreed protocol for site assignment. It is emphasized that the most important drivers for this
are not the impact on management of an individual case, as this is not affected by the
assigned primary site. The importance of uniform primary site assignment is that this should
be evidence-based. Demonstration of the true incidence of tubal origin will indicate the
potential extent to which ovary-conserving fallopian tube-targeted preventative strategies
(including opportunistic salpingectomy) could reduce the incidence, morbidity and mortality
of HGSC. Adopting a uniform approach worldwide will ensure accurate data collection for
comparing disease outcomes in routine practice and clinical trials. This will also promote
wider understanding of the nature of HGSC and the use of standardized pathology protocols
in specimen dissection and reporting.
A uniform approach to site assignment in extrauterine HGSC is recommended by the

International Collaboration on Cancer Reporting (ICCR) (100). In a prospective study
applying these criteria, 83%, 17% and 0% chemonaive cases of HGSC were classified as
tubal, ovarian and peritoneal primaries respectively with a high level of interobserver
agreement (68).

It is essential to implement this protocol worldwide because of the significant variation in
current practice. This move should be preceded by appropriate dissemination of this decision
to all stakeholders including cancer registries as well as healthcare commissioners and
Accepted Article
providers. The term ‘tubo-ovarian HGSC’ is recommended as a diagnostic term, to
distinguish this disease clearly from uterine serous and ovarian low-grade serous carcinomas.
This should take into account all clinical and pathological parameters, including history,
clinical, imaging and surgical findings, and morphology with immunohistochemistry for
WT1, p53 and other markers as appropriate in routine practice. We recommend that the
default categorisation of the majority of cases of extrauterine HGSC as “ovarian” is discarded
in favour of site assignment using the following criteria (101-103) (summarized in Table 4):
 Primary site should be assigned as tubal in the presence of STIC or invasive mucosal
HGSC in either fallopian tube, or when either tube is partly or fully incorporated into
and inseparable from a tubo-ovarian mass.
 Primary site should be assigned as ovarian only when there is ovarian involvement
and the tubes are clearly visible, have been dissected away from the surface of the
ovaries, fully examined by a standardized SEE-FIM protocol and neither STIC nor
invasive mucosal carcinoma is present in either tube.
 Primary site should be assigned as peritoneal only when both tubes and both ovaries
are grossly and microscopically normal; this diagnosis should only be made on cases
undergoing primary surgery and after complete examination of both tubes and both
ovaries using a standard protocol.
 In cases diagnosed on an omental/peritoneal biopsy or a cytological sample and where
primary surgery is not undertaken, the presumed primary site should be assigned as
tubo-ovarian. By the 2014 WHO criteria, PPC is likely to become vanishingly rare,
making it unnecessary to include this as part of the differential diagnosis.
 Chemotherapy alters disease distribution; in most cases there is sufficient disease
remaining to enable categorization using the above criteria, but those with no residual
disease should be assigned as tubo-ovarian.
 In cases where HGSC occurs following previous removal of tubes and ovaries which
were not fully sampled, and uterine origin has been excluded, the presumed primary
site may be assigned as “tubo-ovarian” if salpingo-oophorectomy was recent;
recurrences after STIC have been reported up to 6 years later (104).

 Site assignment as ‘undesignated’ should be avoided as far as possible, and used only
in the rare event that a tumour does not fit into any of the above categories and/or
there remains doubt over whether it is of tubo-ovarian or endometrial origin.
Accepted Article
A practical alternative to primary site assignment as recommended by FIGO and perhaps
a consideration for the future would be to adopt ‘tubo-ovarian’ as the favoured diagnostic
term. In most instances the disease presents at advanced stage with obvious tubal
involvement evident macroscopically. Detailed tubal examination is only necessary in
low-stage cases as there is potential to under-stage if this is not meticulously carried out.
MOLECULAR PATHOLOGY OF HGSC

The Cancer Genome Atlas (TCGA) study, with sequencing of more than 300 tubo-ovarian
HGSC, yielded important insights into the molecular abnormalities underlying this tumour
type (74). It confirmed the ubiquity of TP53 mutations, and also highlighted the fact that
apart from TP53, BRCA1 and BRCA2, there are few recurrent mutations in HGSC, in contrast
to many other tumour types. HGSC is a tumour characterized by aneuploidy and large
numbers of somatic copy number alterations, rather than recurrent mutations. The mechanism
accounting for this phenotype is defects in cellular mechanisms that allow for high-fidelity
repair of DNA double strand (DS) breaks; this leads to mutation prone repair mechanisms,
resulting in aneuploidy/copy number abnormalities. Genetic abnormalities accrue rapidly
within the dividing tumour cells, leading to dramatic intratumoral heterogeneity; in a study of
advanced stage HGSC where tumour from multiple anatomic sites in the same patient was
compared, fewer than 50% of mutations were present in all sampled specimens (75). Thus in
HGSC there is loss of the ability to repair DNA DS breaks as an early event, followed by
accrual of large numbers of copy number abnormalities and inter- and intra-tumoral
heterogeneity.
Both BRCA1 and BRCA2 encode genes that play critical roles in DS DNA repair through the
error-free homologous recombination pathway. In the TCGA series, 33% of tumours had
germline or somatic mutations in either BRCA1 or BRCA2, or BRCA1 promoter methylation,
and an additional 18% of tumours had other mutations e.g. EMSY amplification, that would
also be predicted to result in defective homologous recombination. The BRCA1/2 mutation
frequency may be low in the TCGA for technical reasons; for example, only 9% of their

cases had germline BRCA1 mutations, while other studies have found considerably higher
frequencies, including 17 % in the Australian Ovarian Cancer Study of more than 1000
patients (105), and 26% in our centre (106). Defects in homologous recombination, whether
Accepted Article
as a result of BRCA mutation or other mechanisms, would be expected to confer sensitivity
to PARP inhibitors to the affected tumour cells; at present we do not have a sensitive and
specific biomarker that predicts responsiveness of HGSC to this important new class of drug.
A minority of HGSC have amplification of Cyclin E1 (CCNE1), a molecular abnormality

that is mutually exclusive of BRCA mutation and is associated with a worse
prognosis/platinum resistance, compared to HGSC without CCNE amplification (107-109).
CCNE amplification often co-exists with AKT amplification and treatment directed against
cyclin-dependent kinase-2 (CDK2) and AKT, with small molecule kinase inhibitors, is a
potential approach to targeted treatment of these platinum resistant HGSC (110).
Careful examination of the sequence at DNA break points i.e. sites of DS DNA breaks
repaired by mechanisms other than the high-fidelity homologous recombination pathway, has
revealed two groups of HGSC, based on how the break was repaired (111).In classical non-
homologous end-joining (NHEJ) there is no or minimal (<3 base pairs) sequence homology
at breakpoints, while with micro-homology mediated end joining (MMEJ) there are short (>5
base pairs) sequences of homology flanking the break point. MMEJ is mediated by DNA
polymerase 
mediated repair of DS DNA breaks and this group is associated with a worse prognosis,
compared to the former group. Interestingly, CCNE amplification is a feature of the tumours
with MMEJ mediated repair. A comparison of HGSC with MMEJ versus NHEJ defects is
shown in Table 5. Tumour cells with predominantly MMEJ-mediated DS DNA repair are
candidates for targeted therapy directed against POLQ
PATHOLOGICAL ASSESSMENT OF PROGNOSIS IN HGSC; CHEMOTHERAPY

RESPONSE SCORE
Despite the tremendous genomic diversity of HGSC, the number of recurrent and actionable
mutations associated with this disease is disappointingly low. An overview of the many
prognostic and predictive markers currently under scrutiny in extrauterine HGSC is beyond
the scope of this review. In the near future it is likely that incorporation of targeted

therapeutic agents into clinical practice will mandate the routine reporting of specific
parameters, as already exemplified by demonstration of defective homologous recombination
in DNA repair for evaluating suitability for PARP inhibitors, or in the near future, and in
Accepted Article
common with other solid cancers, immune marker expression for immune checkpoint or
other immune modulatory therapies.
A novel challenge for the pathologist is the consistent and reproducible reporting of response
to classic and novel treatments. The recently defined Chemotherapy Response Score (CRS)
has been found to be prognostically relevant and an adjunct to other indicators of response,
namely serum CA125 and surgical debulking status (112). The system scores the pathological
response to neo-adjuvant chemotherapy (NACT) on interval debulking specimens (IDS). In
this system the omental section with the maximal residual tumour is evaluated. In instances
where the omentum is not removed at IDS, or was not involved prior to NACT, other disease
sites should be descriptively reported. The system has been validated for use in IDS
specimens of HGSC and not other morphological types of EOC. The CRS correlates with
progression free survival, and, to a lesser extent, overall survival. A high score (CRS3) has a
94% negative predictive value for platinum sensitivity. This therefore has high potential for
treatment modulation and serving as an early end-point in clinical trials. The system,
summarised in table 6, has been independently validated (113) and shown to be simple and
easy to apply in routine reporting, as well as being highly reproducible amongst pathologists
regardless of specialist experience (114). The CRS system has been incorporated into the
histology reporting guidelines of the ICCR (100) and College of American Pathologists
(115). An online tutorial has been set up where participants can view scanned examples of
cases showing different chemotherapy response scores, and test their own proficiency in
using the system prior to applying this to their own clinical practice
(http://www.gpecimage.ubc.ca/aperio/images/crs). The system has potential to form the basis
for reporting of the pathological response to other forms of therapy in the future.
CONCLUSIONS
The morbidity and mortality from EOC is largely due to HGSC. It is important for us to fully
understand the molecular biology of this disease and embrace new developments that enable
accurate diagnosis, staging, primary site assignment and treatment response. This will help to
build the strong foundation necessary to underpin future developments in prognostic and
predictive markers in the rapidly changing era of personalised medicine.

Acknowledgement
All authors contributed equally to the contents of this review.
Accepted Article
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Accepted Article
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Accepted Article
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Accepted Article
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Accepted Article
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Figure Legends
Figure 1. HGSC typically shows a variety of patterns which often coexist in the same case.
1a: Typical glandular-papillary pattern;
1b: Micropapillary pattern resembling LGSC;
1c: Glandular pattern resembling EC (inset: aberrant diffuse “mutation-type” p53 staining in
this case);
1d: Microcystic/microglandular pattern;
1e: Solid pattern;
1f: Transitional-like pattern.
Figure 2a. LGSC typically shows a pattern of monotonous delicate papillary structures
surrounded by cleft-like spaces.

Figure 2b: In some cases of LGSC there may be foci showing nuclear enlargement,
prominent nucleoli and appreciable mitotic activity; nuclear pleomorphism and bizarre nuclei
not seen. Such cases may be difficult to distinguish from HGSC particularly in small biopsy
specimens (inset: normal ‘wild-type’ p53 nuclear expression pattern in this case).
Accepted Article
Figure 3a: STIC at fimbrial end of fallopian tube characterized by marked nuclear atypia and
other features in non-ciliated cells.
Figure 3b: STIC showing increase in Ki67 staining.
Figure 3c: STIC showing aberrant totally negative “mutation-type” p53 staining, in presence
of a positive control in background cells.
Figure 3d: STIC, an example showing aberrant diffuse “mutation-type” staining with p53.
Figure 4: In the absence of agreement on uniform categorisation, there is potential for

variation in assignment of primary site in extrauterine HGSC. An example of a case with (1)
STIC, (2) a small invasive ovarian HGSC and (3) large volume peritoneal disease is
illustrated. This could be categorized as ovarian (if the pathologist sees the largest dimension
(a) and this exceeds 5mm) or peritoneal (if the pathologist sees the smallest dimension (b)
and this is less than 5mm) based on traditional criteria, tubal if the newly proposed criteria
are accepted (ref 102), or undesignated if it is felt that there remains doubt on site of origin.
TABLE 1. Low Stage (I/II) Versus High Stage (III/IV) Distribution of Subtypes of
Epithelial Ovarian Cancer (EOC) (7).
Ovarian Carcinoma Percentage (All Percentage (Stage Stage I/II: Stage

Histotype Stages) III/IV) III/IV
High grade serous 68.1% 87.7% 20:80
Clear cell 12.2% 4.5% 82:18
Endometrioid 11.3% 2.5% 88:12
Mucinous 3.4% 1.2% 82:18
Low grade serous 3.4% 5.3% 18:82

Table 2. MARKERS OF VALUE IN TYPING EPITHELIAL OVARIAN CANCERS
(EOCs)
Accepted Article
HGSC LGSC ENDOMETRIOID CLEAR CELL
WT1 Diffuse positive Diffuse positive Negative Negative
p53 Mutation type Wild type Wild type or Wild type

mutation type
p16 Diffuse positive Focal positive Focal positive Focal positive
ER Diffuse or focal Diffuse positive Diffuse positive Negative

positive or
negative
HNF1 beta Negative Negative Negative Diffuse or focal

positive
Napsin A Negative Negative Negative Diffuse or focal

positive
HGSC = High grade serous carcinoma

LGSC = Low grade serous carcinoma
ER = Oestrogen receptor
HNF1 beta = hepatocyte nuclear factor 1 beta
These are the most common staining patterns but exceptions may occur with all of the
markers.
Table 3
p53 immunohistochemistry pattern and interpretation (39)
Pattern p53 IHC TP53 mutation type % in

Interpretation HGSC
TP53 MUTATION ABSENT
Wild type Normal No mutation 0
TP53 MUTATION PRESENT
Overexpression Abnormal Non-synonymous (missense); also in-frame 66%
deletion, splicing
Complete absence/ null Abnormal Indels, stopgains, splicing mutations 25%
Cytoplasmic Abnormal Indels and stopgains with disruption of the 4%
nuclear localization domain
Wild type Normal* Truncating mutation 5%
HGSC- high-grade serous carcinoma

Table 4: CRITERIA FOR ASSIGNMENT OF PRIMARY SITE IN TUBO-
OVARIAN HGSC (102)
Criteria Primary site Comment

Accepted Article
STIC present Fallopian tube Regardless of presence and
size of ovarian and
peritoneal disease
Invasive mucosal Fallopian tube Regardless of presence and
carcinoma in tube, with or size of ovarian and
without STIC peritoneal disease
Fallopian tube partially or Fallopian tube Regardless of presence and
entirely incorporated into size of ovarian and
tubo-ovarian mass peritoneal disease
No STIC or invasive Ovary Both tubes should be
mucosal carcinoma in clearly visible and fully
either tube in presence of examined by a
ovarian mass or standardized SEE-FIM
microscopic ovarian protocol.
involvement
Regardless of presence and
size of peritoneal disease
Both tubes and both Primary peritoneal HGSC As recommended in WHO
ovaries grossly and blue book 2014 (2)
microscopically normal This diagnosis should only
(when examined entirely) be made in specimens
or involved by benign removed at primary
process in presence of surgery prior to any
peritoneal HGSC chemotherapy; see below
for samples following
chemotherapy.
HGSC diagnosed on small Tubo-ovarian Note: this should be
sample, peritoneal/ supported by
omental biopsy or clinicopathological
cytology findings including
immunohistochemistry to
exclude mimics,
principally uterine serous
carcinoma
Post-chemotherapy with As above
residual disease
Post-chemotherapy with Tubo-ovarian
no residual disease

Table 5: Comparison of Characteristics of HGSC with Micro-homology mediated end joining
(MMEJ) versus Non-homologous end joining (NHEJ) repair of double strand DNA breaks
(111)
Accepted Article
HGSC with MMEJ HGSC with NHEJ
CCNE amplification BRCA1/2 mutations
Poor outcome (worse response to Better response to chemotherapy,
platinum- taxane chemotherapy) including some long-term survivors
Target: POLQ (?) Target: HR deficiency (i.e. PARPi)
Table 6: Chemotherapy Response Score: Summary of criteria (112)
CRS score Criteria

CRS1: No or Mainly viable tumour with no or minimal regression-associated fibro-
minimal tumour inflammatory changes* limited to a few foci
response
Note: cases in which it is difficult to decide between regression and
tumour-associated desmoplasia or inflammatory cell infiltration
CRS2: Partial Appreciable tumour response amidst viable tumour, both readily
response identifiable and tumour regularly distributed
Note: cases ranging from multifocal or diffuse regression associated

fibro-inflammatory changes*, with viable tumour in sheets, streaks or
nodules, to extensive regression associated fibro-inflammatory
changes* with multifocal residual tumour which is easily identifiable
CRS3: Total or No residual tumour OR minimal irregularly scattered tumour foci seen
near-total as individual cells, cell groups or nodules up to 2mm in maximum size
response
Note: cases showing mainly regression associated fibro-inflammatory
changes* or, in rare cases, no/very little residual tumour in complete
absence of any inflammatory response; advisable to record whether “no
residual tumour” or “microscopic residual tumour present”
*Regression associated fibro-inflammatory changes: Fibrosis associated with macrophages, including

foam cells, mixed inflammatory cells and psammoma bodies; to distinguish from tumour-related
inflammation or desmoplasia

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DR NAVEENA SINGH (Orcid ID : 0000-0003-1782-1967)

DR GLENN MCCLUGGAGE (Orcid ID : 0000-0001-9178-4370)

TITLE: HIGH GRADE SEROUS CARCINOMA (HGSC) of TUBO-OVARIAN

AUTHORS: Naveena Singh1, W Glenn McCluggage2, C Blake Gilks3

Tel: (44) 2032460217/0198

Key words: High grade serous carcinoma, HGSC, pathology, immunohistochemistry,

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PROBLEMATIC AREAS IN HISTOLOGICAL TYPING OF EOCs

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Distinction between HGSC and LGSC

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Transformation of LGSC into High Grade Carcinoma

Distinction between HGSC and Endometrioid Carcinoma

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Distinction between HGSC and Clear Cell Carcinoma

Transitional Cell Carcinoma Is Removed From The WHO 2014 Classification Of

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MIXED EPITHELIAL OVARIAN CARCINOMAS ARE EXTREMELY

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OVERWHELMING EVIDENCE FOR FALLOPIAN TUBE ORIGIN OF

Traditional Theories of Site of Origin of “Ovarian HGSC”

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Fallopian Tube as Site of Origin of HGSC

(a) Frequency of STIC and Tubal Mucosal Invasive Carcinomas in Sporadic/Non-High

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(b) Clonal Relationship Between STIC and Concurrent HGSC

(c) Evidence that STIC Represents the Primary Lesion

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It is challenging to demonstrate whether or not STIC represents a primary or metastatic lesion

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FALLOPIAN TUBE SAMPLING - WHAT IS THE OPTIMAL PROTOCOL?

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PROPOSAL FOR UNIFORM SITE ASSIGNMENT IN EXTRAUTERINE HGSC

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A uniform approach to site assignment in extrauterine HGSC is recommended by the

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MOLECULAR PATHOLOGY OF HGSC

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A minority of HGSC have amplification of Cyclin E1 (CCNE1), a molecular abnormality

PATHOLOGICAL ASSESSMENT OF PROGNOSIS IN HGSC; CHEMOTHERAPY

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Figure 3b: STIC showing increase in Ki67 staining.

Figure 4: In the absence of agreement on uniform categorisation, there is potential for

Ovarian Carcinoma Percentage (All Percentage (Stage Stage I/II: Stage

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WT1 Diffuse positive Diffuse positive Negative Negative

p53 Mutation type Wild type Wild type or Wild type

p16 Diffuse positive Focal positive Focal positive Focal positive

ER Diffuse or focal Diffuse positive Diffuse positive Negative

HNF1 beta Negative Negative Negative Diffuse or focal