ORIGINAL ARTICLE

Adult Granulosa Cell Tumor With

High-grade Transformation
Report of a Series With FOXL2 Mutation Analysis
Yinka Fashedemi, FRCPath,* Michael Coutts, FRCPath,† Olga Wise, FRCPath,‡
Benjamin Bonhomme, PhD,§ Gavin Baker, MSc,∥ Paul J. Kelly, FRCPath,∥
Isabelle Soubeyran, PhD,§ Mark A. Catherwood, PhD,¶ Sabrina Croce, MD,§
and W. Glenn McCluggage, FRCPath∥

grade component of 2 of the cases. In 1 case, there was diffuse

Abstract: Adult granulosa cell tumor (AGCT) is a low-grade malig-
nant neoplasm with a significant propensity for late recurrence and
metastasis. Almost all AGCTs are composed of cells with bland nu-
clear features and even when these tumors recur or metastasize, the
nuclear features are almost always low-grade. We report 5 cases of
AGCT in patients aged 37 to 88 years composed of areas of typical
AGCT with low-grade morphology admixed with areas of high-grade
morphology, with marked nuclear atypia, often with bizarre multi-
nucleate cells and high mitotic activity; this is the first reported series of
high-grade transformation in AGCTs. The high-grade areas often
morphologically closely resembled juvenile granulosa cell tumor with
abundant eosinophilic cytoplasm, significant mitotic activity, and in-
termediate sized follicles. Four cases were FIGO stage IA at diagnosis
and 1 was stage IIIC with omental involvement. FOXL2 mutation
analysis of both the morphologically low-grade and high-grade areas
in 4 of 5 cases confirmed the presence of missense point mutation,
c.402C > G, p.(Cys134Trp), providing conclusive evidence that the
high-grade component represents transformation of typical AGCT
rather than the coexistence of another sex cord-stromal tumor, such
as juvenile granulosa cell tumor, which has been suggested for
such neoplasms. In 3 of 4 cases where immunohistochemistry was
undertaken, there was a striking difference between the p53 staining in
the low-grade and high-grade components with wild-type staining in
the former and diffuse mutation-type immunoreactivity in the latter,
suggesting that TP53 mutation is likely to play a role in high-grade
transformation. TP53 mutation analysis covering exons 4 to 10 was
undertaken in 4 cases and TP53 mutations were identified in the high-
From the *Department of Cellular Pathology, Basildon University
Hospital, Basildon; †Department of Pathology, Maidstone General
Hospital, Maidstone; ‡Department of Pathology, St Thomas Hospi-
tal, London; Departments of ∥Pathology; ¶Haematology, Belfast
Health and Social Care Trust, Belfast, Northern Ireland, UK; and
§Department of Biopathology, Institut Bergonié, Comprehensive
Cancer Center, Bordeaux, France.
Conflicts of Interest and Source of Funding: The authors have disclosed
that they have no significant relationships with, or financial interest
in, any commercial companies pertaining to this article.
Correspondence: W. Glenn McCluggage, FRCPath, Department of
Pathology, Belfast Health and Social Care Trust, Grosvenor Road,
Belfast BT12 6BA, UK (e-mail: glenn.mccluggage@belfasttrust.hscni.
net).
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.
block-type p16 staining in the high-grade component. Follow-up in
the 4 stage IA neoplasms revealed no evidence of tumor recurrence in
3 (6 to 9 mo follow-up) while the other patient developed mediastinal,
peritoneal, and pulmonary metastasis 17 months after diagnosis. High-
grade transformation is uncommon in AGCTs and given that one of
our cases was advanced stage at diagnosis, another exhibited wide-
spread metastasis within a short period and there have been occasional
case reports of aggressive behavior in AGCTs with high-grade trans-
formation, this event may herald an aggressive clinical course.
Key Words: ovary, adult granulosa cell tumor, FOXL2 mutation,
high-grade transformation, p53
(Am J Surg Pathol 2019;43:1229–1238)
A dult granulosa cell tumor (AGCT) is one of the com-
monest ovarian sex cord-stromal tumors and is regarded
as a low-grade malignant neoplasm with a significant pro-
pensity for late recurrence and metastasis.1 Most AGCTs are
composed of cells with bland nuclear features arranged in a
variety of architectural patterns and even when these tumors
recur or metastasize, the nuclear features are almost always
bland and low-grade.2 There have been occasional case re-
ports of typical AGCT with areas of “high-grade” malignant
morphology, either in the original neoplasm or in the re-
currence; this has generally been referred to as sarcomatous
transformation.3–5 In this study, we report a small series of
AGCTs with high-grade transformation. We undertook an
immunohistochemical and FOXL2 mutation analysis of
both the morphologically low-grade and high-grade areas
and provide evidence that the high-grade component repre-
sents transformation of typical AGCT rather than the co-
existence of another sex cord-stromal tumor, such as juvenile
granulosa cell tumor (JGCT), which has been suggested for
such neoplasms.6 We also show that TP53 mutation is likely
to play a role in the high-grade transformation.
MATERIALS AND METHODS
The cases derived from the pathology archives of the
institutions to which the authors are affiliated together with

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Fashedemi et al
the consultation practice of one of the authors (W.G.M.).
Hematoxylin and eosin and immunohistochemical-stained
slides were reviewed by the authors during the preparation
of this manuscript. All available slides were reviewed; in the
in-house cases, all the slides were available while in the
consultation cases, often only selected slides were available.
Follow-up was obtained from the referring pathologists or
clinicians involved in the management of the patients.
Immunohistochemistry
Immunohistochemistry for p53, MIB1, inhibin and
p16 was carried out on both the low-grade and high-grade
components. Inhibin staining was classified as negative,
focal (< 50%) or diffuse ( ≥ 50%). p16 staining was clas-
sified as negative, non–block-type (patchy positive stain-
ing involving <90% of tumor cells) or block-type
(involving at least 90% and essentially all of the tumor
cells). The MIB1 proliferation index was subjectively as-
sessed in areas which exhibited the highest staining. p53
staining was classified as mutation-type (diffuse strong
positive staining in > 80% of cells or completely negative)
or wild-type (heterogenous staining of variable intensity in
<80% of cells).7,8
FOXL2 Mutation Methods
FOXL2 mutation analysis was undertaken in 4 cases
(cases 2, 3, 4, 5). A hematoxylin and eosin–stained section
cut from each formalin-fixed paraffin-embedded (FFPE)
block was reviewed by pathologists involved in the study
and tumor tissue from both the low-grade and high-grade
components was selected for analysis. In most cases, the
low-grade and high-grade components were present in
different blocks but sometimes the 2 components were in
the same paraffin block requiring microdissection. Tumor
tissue from the blocks was scraped off with coring (1 mm
diameter) with a uni-core (Sigma-Aldrich, St Louis, MO)
or slide scraping. After an overnight proteinase K diges-
tion step at 70°C, DNA extraction was performed with
Maxwell RSC DNA FFPE kit according to the manu-
facturer’s protocols on a Maxwell RSC device from
Promega (Promega, Madison, WY). DNA was quantified
using QuantiFluor ONE dsDNA System (Promega).
The detection of mutations within exon 1 of the
FOXL2 gene was performed using Sanger sequencing. A
part of FOXL2 exon 1 (the amplicon size was 182 pb) was
amplified using the following primers: FOXL2 forward
primer 5′-CACAACCTCAGCCTCAACGAGTGC-3′
and FOXL2 reverse primer 5′-TTGCCGGGCTGGA
AGTGCG-3′. Briefly, 50 ng of DNA was amplified with
the thermal cycling profile of 95°C for 10 minutes, then 35
cycles of 95°C for 30 seconds, 68°C for 45 seconds, 72°C
for 45 seconds, with a final extension at 72°C for 10 mi-
nutes. Direct sequencing was performed with the above
primers using the Big-Dye DyeDeoxy terminator cycle
sequencing kit (Applied Biosystems, Foster City, CA).
Sequencing reactions were carried out on the ABI Prism
3100 Genetic Analyzer (Applied Biosystems). As a refer-
ence for the FOXL2 gene, NM_023067.3 aligned on a
sequence of chromosome 3 (hg19) was used.
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Am J Surg Pathol  Volume 43, Number 9, September 2019
TP53 Mutation Methods
TP53 mutation analysis was undertaken in 4 cases
(cases 2, 3, 4, 5). DNA was extracted from four 10 μm
sections from each case from both the low-grade and
high-grade components, which were selected as de-
scribed in the FOXL2 mutation methods. Standard
procedures were used according to the manufacturer’s
protocol for DNA extraction (QIAampDNA FFPE
Tissue Kit; Qiagen, Manchester, UK), except that in-
cubation at 56°C was extended to 3 days to optimize the
purification of genomic DNA. Quantification of DNA
was performed by an absorbance method using the
NanoDrop 2000c spectrophotometer. Exons 4 to 10
were amplified with primers as previously described.9
For each exon, 100 ng of DNA was amplified by pol-
ymerase chain reaction (PCR) in a 25-μL reaction
mixture containing 3 mM MgCl2, 200 μM dNTP, 1 U of
Platinum Taq (Invitrogen, Paisley, UK) and 10 μM of
each primer. Cycling conditions were as follows: 94°C
for 10 minutes, 40 cycles at 94°C for 1 minute, 55°C for
1 minute, and 72°C for 1 minute with a final extension at
72°C for 10 minutes. PCR products were directly se-
quenced by Sanger sequencing with Big-Dye termi-
nators version 3.1 (Applied Biosystems), using both
forward and reverse primers. Mutations were confirmed
by amplifying a duplicated PCR product. Poly-
morphism status was assessed and the presence of mu-
tations were confirmed with reference to the IARC
TP53 database.
RESULTS
Clinical Features
Brief clinical and pathologic features of the 5 cases
are presented in Table 1. The patients ranged in age from
37 to 88 years and all presented with symptoms related to
the presence of an ovarian mass. All cases were unilateral;
2 involved the right ovary and 2 the left. In the other case
(case 4), a large right adnexal mass was present with
omental and sigmoid colon metastases but the ovary was
never removed; the patient underwent a small intestinal
bypass and omental resection and the diagnosis was made
on the omental resection specimen. Four cases were FIGO
stage IA at diagnoses and 1 (case 4) was stage IIIC with
omental and sigmoid involvement. Follow-up in the 4
stage IA neoplasms revealed no evidence of tumor
recurrence in 3 (6 to 9 mo follow-up) while the other
developed mediastinal, peritoneal, and pulmonary
metastasis 17 months after diagnosis. The patient with
stage IIIC disease was not considered fit enough for any
further treatment and was transferred to palliative care
and lost to follow-up.
Pathologic Features
The tumors ranged in size from 9 to 28 cm in max-
imum dimension. Where the gross description was avail-
able, the tumors were described as predominantly solid
with minor cystic foci and with a yellow/gray cut surface.
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Am J Surg Pathol  Volume 43, Number 9, September 2019 High-Grade Adult Garanulosa Cell Tumour

TABLE 1. Clinicopathologic Features of Cases Included in Study

Site of
Tumor Metastatic
Case Age Clinical Size Adjuvant FIGO Disease at
No. (y) Tumor site Presentation Operative Procedure (cm) Treatment Stage Diagnosis Follow-up
1 59 Right ovary 48 h history of Hysterectomy and BSO 16.5 Carboplatin, IA None Mediastinal, peritoneal and
acute ifosfamide pulmonary metastasis 17
abdominal and mo after diagnosis; alive
pain etoposide with disease 19 mo after
chemother- diagnosis
apy
2 51 Right ovary Pelvic pain and Hysterectomy, BSO, 21 None IA None No evidence of recurrence
bloating omentectomy, pelvic at 9 mo
lymphadenectomy
3 37 Left ovary 6 wk history of Hysteroscopy, left 9 None IA None No evidence of recurrence
PV bleeding. salpingo- at 6 mo
Left adnexal oophorectomy, omental
mass identified biopsy, pelvic and para-
on imaging aortic lymph node
sampling
4 87 Large right Intestinal Small intestinal bypass NA None IIIC Sigmoid Referred to palliative case
adnexal obstruction and omental resection colon and and then lost to follow-up
mass but omentum
ovary
never
removed
5 52 Left ovary Found to have Hysterectomy, BSO, 28 None IA None No evidence of recurrence
large ovarian omentectomy at 6 mo
mass on
imaging
BSO indicates bilateral salpingo-oophorectomy; NA, not available.

The omentum in case 4 measured 30 cm in maximum di-
mension and was almost totally involved by tumor.
Histologically all the tumors had areas of typical
AGCT with low-grade cytologic features and scant cyto-
plasm. In these areas, tumor cells with angulated nuclei,
some containing grooves, and scant cytoplasm were ar-
ranged in various architectural patterns, including solid,
nested, and corded/trabecular. Microfollicles and Call-
Exner bodies were identified in some cases. The mitotic
count per 10 high-power fields in these low-grade areas
was 30, 1, 6, 1, and 1 in cases 1 to 5, respectively. Necrosis
was not identified in these areas, except in 1 case (case 2)
where there were areas of infarct type necrosis.
In all cases, there was an abrupt transition between
the low-grade areas and foci where the tumor cells ex-
hibited marked nuclear atypia, often with bizarre multi-
nucleate forms. In these foci, the tumor cells were
predominantly arranged in diffuse sheets but in some
cases, there was a focal nested and/or corded/trabecular
architecture. The tumor cells in these morphologically
high-grade areas had abundant eosinophilic, and some-
times focally clear, cytoplasm. In cases 1, 2, and 3, inter-
mediate sized follicles, some containing eosinophilic
material, were present. The mitotic count per 10 high-
power fields in these areas was 50, 3, 6, 4, and 1 in cases 1
to 5, respectively, and atypical mitoses were identified in
some cases. Areas of necrosis were typically present.
In all cases, there was a substantial amount of both
low-grade and high-grade components within the tumor.
Figures 1–3 illustrate representative examples of the
neoplasms.
Immunohistochemical Results
The immunohistochemical results within the low-
grade and high-grade components are summarized in
Table 2. Typically the Ki67 (MIB1) proliferation index
was very low in the low-grade areas (1%) and markedly
elevated in the high-grade areas (up to 50%), although
there was 1 case (case 5) where the MIB1 proliferation
index was only 1% in the high-grade foci; in this case, the
mitotic count was also low in the high-grade areas but
there confluent areas of marked nuclear atypia with tumor
cell necrosis and diffuse mutation-type p53 staining (see
below). There was no obvious difference in the degree of
inhibin staining between the low-grade and high-grade
components; in 3 cases, the degree of positive staining was
similar in both components while in 1 case each it was
higher in the low-grade or high-grade component. In 3 of 4
cases tested, p16 exhibited non–block-type immunor-
eactivity in the low-grade and high-grade foci, although
there was generally more widespread staining in the high-
grade foci. In the other case, the high-grade component
exhibited block-type immunoreactivity with strong nu-
clear and cytoplasmic staining of all tumor cells while the
low-grade component exhibited non–block-type im-
munoreactivity. In 3 of 4 cases where p53 staining was
undertaken, the immunohistochemical staining pattern
was different in the low-grade and high-grade areas.

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Fashedemi et al Am J Surg Pathol  Volume 43, Number 9, September 2019

FIGURE 1. Representative areas of AGCT with typical low-grade morphology from the various cases. Various architectural patterns
are present, including trabecular, diffuse and microfollicular (A–D).

In these cases, the low-grade areas exhibited wild-type
staining with scattered positive nuclei while the high-grade
areas exhibited diffuse mutation-type immunoreactivity.
In the other case, both components exhibited wild-type
immunoreactivity.
Figures 4 and 5 illustrate representative examples of
the immunohistochemistry.
FOXL2 Mutation Results
All cases where molecular testing was undertaken
harbored the missense point mutation, c.402C > G, p.
(Cys134Trp), which is characteristic of AGCT; the same
mutation was present in the low-grade and high-grade
components (Fig. 6).
TP53 Mutation Results
TP53 mutations were detected in the high-grade
components of cases 2 and 3. A missense mutation
(c.659A > C, p.Y220S) was detected in exon 6 of case 2
and a nonsense mutation (c.916C > T, p.R306*) in exon 8
of case 3. No mutations were detected in the low-grade
components in cases 2 and 3 or the low-grade or high-
grade components in cases 4 and 5.
DISCUSSION
AGCT is the most common malignant ovarian sex
cord-stromal tumor. It is considered a low-grade malig-
nant neoplasm with a significant propensity for late re-
currence and metastasis, often in excess of 10 years after
diagnosis.1,2 Some patients develop multiple tumor re-
currences over a period of many years. While the clinical
course is often protracted, most patients who develop tu-
mor recurrence will eventually die of the neoplasm. Al-
most all AGCTs are composed of cells with bland nuclear
features arranged in a variety of architectural patterns and
even when these tumors recur or metastasize, the nuclear
features are almost always bland and low-grade.2 In this
study, we report 5 AGCTs exhibiting an abrupt transition
from areas of typical morphologically low-grade neoplasm
to areas of high-grade tumor. In reporting these unusual
cases, we compare the immunophenotype and FOXL2

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Am J Surg Pathol  Volume 43, Number 9, September 2019 High-Grade Adult Garanulosa Cell Tumour

FIGURE 2. Representative areas of AGCT with high-grade morphology. There is a sharp demarcation between a morphologically
low-grade area (bottom left) and a high-grade area (top right) (A). High-grade area with marked nuclear atypia, including
multinucleate cells (B). High-grade area where the tumor cells have abundant eosinophilic cytoplasm (C). High-grade areas with
bizarre nuclear atypia and abundant mitotic activity, including atypical mitoses (D). A—case 3, B and C—case 2, D—case 3.

mutation status between the 2 components and discuss the
underlying mechanisms behind the high-grade trans-
formation.
In all cases, the low-grade areas were morphologi-
cally typical of AGCT with a variety of architectural
patterns. The high-grade areas were composed of mark-
edly atypical bizarre cells, often with multinucleate cells
and areas of necrosis. There was usually significant mitotic
activity, often with atypical mitoses. In 1 case (case 5), the
mitotic count was low in the high-grade areas and similar
to that in the low-grade areas. In this case, large confluent
areas of markedly atypical cells were present with tumor
cell necrosis and there was diffuse mutation-type staining
with p53 (discussed below). We make the point that, in our
experience, it is not rare to find foci in AGCT where the
nuclei are slightly larger and where there is more mitotic
activity than is typically seen in these neoplasms. We have
not included such cases in our study which is restricted to
tumors with obvious high-grade malignant appearing foci.
To our knowledge, this is the first series reporting high-
grade transformation within AGCT, although there have
been occasional case reports of “sarcomatous” change
with marked nuclear atypia in these neoplasms.3–5 These
may represent similar tumors to those we report.
Two other points which are worth mentioning are
that luteinized AGCTs may contain cells with more
atypical nuclei than in usual in AGCTs 10,11 and that small
collections of cells with bizarre nuclei, but without sig-
nificant mitotic activity, may occur in AGCTs.12–14 Lu-
teinized AGCT is an uncommon variant of AGCT in
which the tumor cells have abundant eosinophilic or clear
cytoplasm, in contrast to the usual scant cytoplasm of
typical AGCT.10,11 It has arbitrarily been recommended
that at least 50% of the tumor cells should be luteinized
before rendering a diagnosis of luteinized AGCT.10 Lu-
teinized AGCTs are characterized by slightly enlarged
nuclei and a relative lack of nuclear grooves, as compared
with typical AGCT. Although the high-grade areas in our

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Fashedemi et al Am J Surg Pathol  Volume 43, Number 9, September 2019

FIGURE 3. High-grade areas resembling JGCT with tumor cells with abundant eosinophilic cytoplasm forming follicles, some
containing eosinophilic fluid (A–D). A and B, Case 2. C and D, Case 3.

cases had abundant eosinophilic cytoplasm, the nuclear
features were much more atypical than in seen in lutei-
nized AGCTs. Small collections of cells with bizarre nu-
clei, but without significant mitotic activity, may occur in
various sex cord-stromal tumors.12–14 The bizarre nuclei
are often in the form of multinucleate giant cells, some-
times of floret type. They may be seen in Sertoli-Leydig
cell tumors, AGCTs, thecomas, fibromas, and other sex
cord-stromal tumors and can be regarded as analogous to
bizarre/symplastic cells in uterine leiomyomas. They are
probably degenerative in nature and are not thought to be
of any clinical significance, although this is based on fol-
low-up in a limited number of cases. As discussed, in one
of our cases the mitotic count was low and similar between
the low-grade and high-grade components raising the
possibility of symplastic change; however, given the large

TABLE 2. Immunohistochemical Findings

MIB1 Low- MIB1 High- p53 Low- p53 High-grade Inhibin Low- Inhibin High- p16 Low- p16 High-
Case No. grade Areas grade Areas grade Areas Areas grade Areas grade Areas grade Areas grade Areas
1 Not done Not done Not done Not done Diffuse Diffuse Not done Not done
2 1% 10% Wild-type Diffuse Diffuse Diffuse Focal Diffuse
mutation-type
3 1% 30% Wild-type Diffuse Focal Focal Focal Diffuse
mutation-type
4 1% 50% Wild-type Wild-type Diffuse Focal Focal Focal
5 1% 1% Wild-type Diffuse Focal Diffuse Focal Diffuse
mutation-type

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Am J Surg Pathol  Volume 43, Number 9, September 2019 High-Grade Adult Garanulosa Cell Tumour

FIGURE 4. Case 3. MIB1 stain showing low proliferation index in low-grade area (right) and high proliferation index in high-grade
area (left) (A). p16 stain showing largely negative staining in low-grade area (right) and diffuse staining in high-grade area (B).

confluent areas of markedly atypical cells with tumor cell
necrosis and diffuse mutation-type staining with p53, we
excluded symplastic change as an explanation for this
morphology.
In 3 of our cases, the morphology of the high-grade
areas strongly raised the possibility of JGCT due to the
significant nuclear atypia, obvious mitotic activity, abun-
dant eosinophilic cytoplasm, and the presence of inter-
mediate sized follicles. We only identified a single reported
case of combined AGCT and JGCT in the literature; this
occurred in a 12-year-old girl and the tumor was reported
to contain areas of AGCT, JGCT (with areas of marked
atypia) and Sertoli cell tumor.6 In spite of the paucity of
reports, many authorities believe that combined AGCT
and JGCT exists (W. Glenn McCluggage, personal oral
communication). However, given that the underlying
molecular events differ between AGCT and JGCT, we
believe it is likely that mixed tumors with components of
both are extremely uncommon if they exist at all; it is
likely that neoplasms reported as mixed AGCT and JGCT
represent a single neoplastic type with morphologic
mimicry of the other.
Recent studies have shown that about 95% of
AGCTs harbor the c.402C > G, p.(Cys134Trp) mutation
in FOXL2 15–21 while in-frame duplications affecting the
pleckstrin-homology domain of AKT1 and/or activating
AKT1 mutations were identified in 14 of 16 (87.5%)
JGCTs in one study.22 FOXL2 mutation is uncommon in
ovarian sex cord-stromal tumors other than AGCT, al-
though it has been demonstrated in occasional thecomas
and JGCTs.15–21 The mutation was first identified in 90 of
93 (97%) AGCTs in a study in 2009;15 in that study, 3 of

FIGURE 5. p53 stain showing sharp demarcation between wild-type staining in low-grade area (top) and diffuse mutation-type
immunoreactivity in high-grade area (bottom) (A) (case 5). Another case (case 2) showing sharp demarcation between wild-type
staining in low-grade area (left) and diffuse mutation-type immunoreactivity in high-grade area (right) (B).

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Fashedemi et al
FIGURE 6. Electropherogram of one of the cases. Note the presence of the FOXL2 missense point mutation c.402C > G, p.
(Cys134Trp).
14 thecomas (21%) and 1 of 10 JGCTs (10%) also har-
bored the mutation. Mutations were not found in 49 other
ovarian sex cord-stromal tumors or in an additional 329
neoplasms comprising ovarian tumors of non–sex cord-
stromal lineage or breast carcinomas. In another study, 52
of 56 (93%) AGCTs harbored FOXL2 mutation; mor-
phologic reappraisal of the 4 cases exhibiting wild-type
FOXL2 sequences suggested that they may have been
misclassified at diagnosis.17 Another study found FOXL2
mutations in 18 of 20 (90%) AGCTs but not in JGCTs.16
It may be that the small percentage of JGCTs exhibiting
FOXL2 mutation in some studies represent misclassified
AGCTs, perhaps similar to the cases we report. Recently,
Yanagida et al23 demonstrated that heterozygous FOXL2
mutation is persistent in paired primary and recurrent
AGCT highlighting the pathogenic and driver role of
FOXL2 mutation in AGCT tumorigenesis. In the 4 cases
we report where mutational analysis was undertaken,
FOXL2 was mutated in both the low-grade and high-
grade components providing convincing evidence that
these represent AGCTs with high-grade transformation
rather than mixed neoplasms.
A somewhat analogous scenario to what we report is
that some ovarian gynandroblastomas (mixed sex cord-
stromal tumors) are composed of admixtures of Sertoli-
Leydig cell tumor and JGCT and that neoplasms with
this admixture of tumor types often exhibit DICER1
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Am J Surg Pathol  Volume 43, Number 9, September 2019
mutations,24 a molecular hallmark of Sertoli-Leydig cell
tumors.25–28 This suggests that gynandroblastomas com-
posed of Sertoli-Leydig cell tumor and JGCT represent
morphological variants of SLCT.24 This is likely another
example of morphologic mimicry of one tumor type by
another. A small number of ovarian gynandroblastomas
have been shown to harbor FOXL2 mutations28 suggest-
ing that they represent AGCTs with foci resembling other
sex cord-stromal tumors, another example of morpho-
logical mimicry. These points illustrate the propensity for
significant morphologic overlap between the various sex
cord-stromal tumors and the value of molecular testing in
problematic cases where the morphology is “mixed,”
ambiguous or not entirely typical; in such problematic sex
cord-stromal tumors, immunohistochemistry will not help
in differentiating between the various neoplasms.
We compared the immunophenotype between the
low-grade and high-grade areas in our cases. There was no
obvious difference in the expression of inhibin, the most
commonly used sex cord marker, between the low-grade
and high-grade components. In all but 1 case where
immunohistochemistry was undertaken, the MIB1 pro-
liferation index was, as expected, elevated in the high-
grade as compared with the low-grade areas. Stewart et al2
compared the Ki67 (MIB1) proliferation indices in 14
cases of paired primary and recurrent/metastatic AGCT
and found no differences; in all of these cases, the
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Am J Surg Pathol  Volume 43, Number 9, September 2019
cytologic features were low-grade in the primary and re-
current neoplasms with no evidence of high-grade trans-
formation. In our study, there was generally increased
expression of p16 in the high-grade compared with the
low-grade areas. In 1 of our cases, the high-grade com-
ponent exhibited block-type p16 immunoreactivity with
strong nuclear and cytoplasmic staining of all tumor cells.
This is an interesting phenomenon and analogous to what
occurs in some mesenchymal neoplasms. For example,
p16 expression is typically markedly elevated in uterine
leiomyosarcomas, as compared with leiomyomas29–31 and
there is markedly increased expression in areas of atypia
or sarcomatous transformation in cellular angiofibromas,
a mesenchymal neoplasm occurring in the vulvovaginal
and inguino-scrotal regions.32 Our study adds to the list of
gynecological neoplasms which may exhibit block-type
p16 immunoreactivity.
In 3 of 4 cases where immunohistochemistry was
performed, the p53 staining pattern was different in the
low-grade and high-grade areas. In these cases, the low-
grade areas exhibited wild-type staining with scattered
positive nuclei while the high-grade areas exhibited diffuse
mutation-type immunoreactivity. This strongly suggests
that TP53 mutation is important in high-grade trans-
formation in AGCTs. In support of this, we identified
TP53 mutations in the high-grade component in 2 of our 4
cases where mutation testing was performed. Our muta-
tion testing only covered exons 4 to 10. Greater than 90%
of TP53 mutations will be identified by our methods as
most mutations are concentrated in the DNA binding
domain. However, it is recognized that mutations can
occur outside of this region.
The high-grade component in the cases we report
could potentially be confused with a wide range of other
neoplasms, especially if only this component is biopsied or
represented. Awareness of the fact that AGCT may un-
dergo high-grade transformation is crucial to the diagnosis.
We will not discuss the differential diagnosis in detail but
mention that as high-grade foci in AGCT may be positive
with WT1 and exhibit mutation-type staining with p53 and
block-type immunoreactivity with p16, a high-grade serous
carcinoma might enter into the differential diagnosis. In
such cases, the demonstration of positivity with sex cord
markers, such as inhibin, and of a FOXL2 mutation will
assist in establishing a correct diagnosis.
In all cases, except case 4, the tumors were FIGO
stage IA at diagnosis. In case 4, the tumor was stage IIIC
with sigmoid colon and omental metastasis. The ovaries
were not removed in this case but a large right adnexal
mass was present and it was assumed that the ovary was
the primary site. We only have short follow-up (ranging
from 6 to 9 mo) in 3 of our stage IA cases with no evidence
of tumor recurrence. The other stage IA case exhibited
widespread metastatic disease 17 months after diagnosis.
There have also been occasional case reports of “sar-
comatous” change with marked nuclear atypia in AGCTs
which have exhibited an aggressive clinical course.3–5 In 1
of these cases, the initial ovarian tumor comprised a typ-
ical AGCT. The tumor recurred 3 times over a period of
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Copyright r 2019 Wolters Kluwer Health, Inc. All rights reserved.
High-Grade Adult Garanulosa Cell Tumour
92 months and the third recurrence exhibited features of
“sarcomatous change.”3 Two other reported cases of
AGCT which had undergone “sarcomatous change” ex-
hibited aggressive behavior with a rapid fatal course.4,5
This suggests that high-grade transformation in AGCTs
may herald an aggressive clinical course and we suggest
that adjuvant therapy should be considered for such ne-
oplasms, particularly when the tumor has spread beyond
the ovary at diagnosis and even when the neoplasm is
confined to the ovary.
In summary, we report a small series of AGCTs with
high-grade transformation. FOXL2 mutation analysis sug-
gests that the high-grade component represents trans-
formation of typical AGCT rather than the coexistence of
another sex cord-stromal tumor, such as JGCT, which has
been suggested for such neoplasms. TP53 mutation is likely
to play a role in the transformation from typical low-grade
AGCT to a high-grade neoplasm. This high-grade trans-
formation may herald an aggressive clinical course.
REFERENCES
1. Miller K, McCluggage WG. Prognostic factors in ovarian adult
granulosa cell tumour. J Clin Pathol. 2008;61:881–884.
2. Stewart CJ, Brennan BA, Crook ML, et al. Comparison of
proliferation indices in primary adult-type granulosa cell tumors of
the ovary and their corresponding metastases: an analysis of 14 cases.
Int J Gynecol Pathol. 2009;28:423–431.
3. McNeilage J, Alexiadis M, Susil BJ, et al. Molecular characterization
of sarcomatous change in a granulosa cell tumor. Int J Gynecol
Cancer. 2007;17:398–406.
4. Susil BJ, Sumithran E. Sarcomatous change in granulosa cell tumor.
Hum Pathol. 1987;18:397–399.
5. Sonoyama A, Kanda M, Ojima Y, et al. Aggressive granulosa cell
tumor of the ovary with rapid recurrence: a case report and review of
the literature. Kobe J Med Sci. 2016;61:E109–E114.
6. Jarzembowski JA, Lieberman RW. Pediatric sex cord-stromal tumor
with composite morphology: a case report. Pediatr Dev Pathol. 2005;
8:680–684.
7. Köbel M, Ronnett BM, Singh N, et al. Interpretation of p53
immunohistochemistry in endometrial carcinomas: toward increased
reproducibility. Int J Gynecol Pathol. 2019;38(suppl 1):S123–S131.
8. Köbel M, Piskorz AM, Lee S, et al. Optimized p53 immunohis-
tochemistry is an accurate predictor of TP53 mutation in ovarian
carcinoma. J Pathol Clin Res. 2016;2:247–258.
9. Boyle DP, McArt DG, Irwin G, et al. The prognostic significance of
the aberrant extremes of p53 immunophenotypes on breast cancer.
Histopathology. 2014;65:340–352.
10. Ganesan R, Hirschowitz L, Baltrušaitytė I, et al. Luteinized adult
granulosa cell tumor—a series of 9 cases: revisiting a rare variant
of adult granulosa cell tumor. Int J Gynecol Pathol. 2011;30:
452–459.
11. Young RH, Oliva E, Scully RE. Luteinized adult granulosa cell
tumors of the ovary: a report of four cases. Int J Gynecol Pathol.
1994;13:302–310.
12. Young RH, Scully RE. Ovarian sex cord-stromal tumors with
bizarre nuclei: a clinicopathologic analysis of 17 cases. Int J Gynecol
Pathol. 1983;1:325–335.
13. Gaffey MJ, Frierson HF Jr, et al. Ovarian granulosa cell tumors with
bizarre nuclei: an immunohistochemical analysis with fluorescence
in situ hybridization documenting trisomy 12 in the bizarre
component. Mod Pathol. 1996;9:308–315.
14. Young RH. Ovarian sex cord stromal tumours and their mimics.
Pathology. 2018;50:5–15.
15. Shah SP, Köbel M, Senz J, et al. Mutation of FOXL2 in granulosa-
cell tumors of the ovary. N Eng J Med. 2009;25:2719–2729.
16. Kim T, Sung CO, Song SY, et al. FOXL2 mutation in granulosa-cell
tumours of the ovary. Histopathology. 2010;56:408–410.
www.ajsp.com | 1237
Fashedemi et al
17. Jamieson S, Butzow R, Andersson N, et al. The FOXL2 C134W
mutation is characteristic of adult granulosa cell tumors of the ovary.
Mod Pathol. 2010;23:1477–1485.
18. D’Angelo E, Mozos A, Nakayama D, et al. Prognostic significance
of FOXL2 mutation and mRNA expression in adult and juvenile
granulosa cell tumors of the ovary. Mod Pathol. 2011;24:1360–1367.
19. Kim MS, Hur SY, Yoo NJ, et al. Mutational analysis of FOXL2
codon 134 in granulosa cell tumour of ovary and other human
cancers. J Pathol. 2010;221:147–152.
20. Köbel M, Gilks CB, Huntsman DG. Adult-type granulosa cell
tumors and FOXL2 mutation. Cancer Res. 2009;69:9160–9162.
21. Kommoss S, Anglesio M, Mackenzie R, et al. FOXL2 molecular
testing in ovarian neoplasms: diagnostic approach and procedural
guidelines. Mod Pathol. 2013;26:860–867.
22. Bessiere L, Todeschini AL, Auguste A, et al. A hot-spot of in-frame
duplications activates the oncoprotein AKT1 in juvenile granulosa
cell tumors. EBioMedicine. 2015;2:421–431.
23. Yanagida S, Anglesio MS, Nazeran TM, et al. Clinical and genetic
analysis of recurrent adult-type granulosa cell tumor of the ovary:
persistent preservation of heterozygous c.402C > G FOXL2 muta-
tion. PLoS One. 2017;8:12.
24. Wang Y, Karnezis AN, Magrill J, et al. DICER1 hot-spot mutations
in ovarian gynandroblastoma. Histopathology. 2018;73:306–313.
25. Witkowski L, Mattina J, Schonberger S, et al. DICER1 hotspot mutations
in non-epithelial gonadal tumours. Br J Cancer. 2013;109:2744–2750.
1238 | www.ajsp.com
Copyright r 2019 Wolters Kluwer Health, Inc. All rights reserved.
Am J Surg Pathol  Volume 43, Number 9, September 2019
26. Anglesio MS, Wang Y, Yang W, et al. Cancer-associated somatic
DICER1 hotspot mutations cause defective miRNA processing
and reverse-strand expression bias to predominantly mature
3p strands through loss of 5p strand cleavage. J Pathol. 2013;
229:400–409.
27. Heravi-Moussavi A, Anglesio MS, Cheng SW, et al. Recurrent
somatic DICER1 mutations in nonepithelial ovarian cancers. N Eng
J Med. 2012;366:234–242.
28. Conlon N, Schultheis AM, Piscuoglio S, et al. A survey of DICER1
hotspot mutations in ovarian and testicular sex cord-stromal tumors.
Mod Pathol. 2015;28:1603–1612.
29. O’Neill CJ, McBride HA, Connolly LE, et al. Uterine leiomyosar-
comas are characterized by high p16, p53 and MIB1 expression in
comparison with usual leiomyomas, leiomyoma variants and smooth
muscle tumours of uncertain malignant potential. Histopathology.
2007;50:851–858.
30. Ip PP, Lim D, Cheung ANY, et al. Immunoexpression of p16 in
uterine leiomyomas with infarct-type necrosis: an analysis of 35
cases. Histopathology. 2017;71:743–750.
31. Chen L, Yang B. Immunohistochemical analysis of p16, p53, and Ki-
67 expression in uterine smooth muscle tumors. Int J Gynecol Pathol.
2008;27:326–332.
32. Chen E, Fletcher CD. Cellular angiofibroma with atypia or
sarcomatous transformation: clinicopathologic analysis of 13 cases.
Am J Surg Pathol. 2010;34:707–714.
Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.