R E S E A R C H Open Access
Cyclin D1 and PRAME expression in
distinguishing melanoma in situ from
benign melanocytic proliferation of the nail
Young Jae Kim
, Chang Jin Jung
, Hyoungmin Na
, Woo Jin Lee
, Sung Eun Chang
, Mi Woo Lee
, Youngkyoung Lim
and Chong Hyun Won
Background: Distinguishing benign lesion from early malignancy in melanocytic lesions of the nail unit still
remains a diagnostic challenge, both clinically and histopathologically. While several immunohistochemistry (IHC)
stainings have been suggested to help discriminate benign subungual melanocytic proliferation (SMP) and
subungual melanoma in situ (MIS), the diagnostic utility of IHC staining for cyclin D1 and PRAME has not been
thoroughly investigated in melanocytic lesions of nail unit.
Methods: This retrospective study included cases of benign SMP and subungual MIS confirmed by biopsy at Asan
Medical Center from January 2016 to December 2020. Cases of melanocytic activation without proliferation and
melanoma where dermal invasion was identified were excluded. Cyclin D1 and PRAME expression was assessed by
counting proportion of melanocytes with nuclear positivity under 200x magnification.
Results: A total of 14 patients with benign SMP and 13 patients with subungual MIS were included in this study. 11
patients with benign SMP (71.4%) and 5 patients with subungual MIS (38.5%) showed > 60% nuclear
immunostaining for cyclin D1, respectively. While 13 patients with benign SMP (92.9%) showed totally negative
staining for PRAME, 10 patients with subungual MIS (76.9%) exhibited > 50% nuclear immunostaining for PRAME.
Using the cutoff of 10%, PRAME exhibited good overall discrimination between benign SMP and subungual MIS
(AUC = 0.849, 95% CI = 0.659–0.957).
Conclusions: This study suggests that PRAME IHC staining as a reliable discriminator in distinguishing subungual
MIS from benign SMP.
Keywords: Cyclin D1, Melanoma, Nail, PRAME, Retrospective study
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* Correspondence: email@example.com;firstname.lastname@example.org
Young Jae Kim and Chang Jin Jung contributed equally as first authors.
Youngkyoung Lim and Chong Hyun Won contributed equally as
Department of Dermatology, Asan Medical Center, University of Ulsan
College of Medicine, 88 Olympic- ro 43 gil, Songpa-gu, Seoul, Korea
Full list of author information is available at the end of the article
Kim et al. Diagnostic Pathology (2022) 17:41
It still remains a diagnostic challenge to distinguish be-
nign lesion from early malignancy in melanocytic lesions
of the nail unit, both clinically and histopathologically
[1,2]. Although non-invasive diagnostic tools including
the “ABCDEF rule”had been proposed for early clinical
detection of subungual melanoma , still the biopsy
and histopathological evaluation is the gold standard in
distinguishing subungual melanoma in situ (MIS) from
benign subungual melanocytic proliferation (SMP) in-
cluding lentigo and melanocytic nevus . Especially,
assessing melanocyte density along the dermoepidermal
junction of nail unit has been proposed to be a reliable
diagnostic tool to distinguish subungual melanoma in
situ from subungual benign melanocytic proliferation:
melanocyte density more than 30 cells per 1 mm is
highly suggestive of subungual MIS . However, it is
occasionally difficult to histopathologically distinguish
one from another, as melanocyte density can differ sig-
nificantly in subungual MIS and benign SMP depending
on the skin type of the patient and the sampled area .
Immunohistochemistry (IHC) can be used to help in
the diagnostic dilemma of benign melanocytic lesion
versus melanoma in situ. However, HMB-45, highlight-
ing the maturation of melanocyte in benign melanocytic
nevus, may not be useful in the diagnosis of MIS where
the dermal invasion is absent . Meanwhile, p16, serv-
ing as a tumor suppressor protein in the regulation of
cell cycle and senescence, has been suggested to be inac-
tivated or lost in melanoma . Nevertheless, Chu et al.
 found that IHC staining for p16 could not distinguish
subungual lentigo from subungual MIS.
Cyclin D1, a protein encoded by CCND1 gene, is one of
the key components of physiologic regulation of cell cycle.
Cyclin D1 regarded as oncogenic protein to promote cell
proliferation has been reported to be upregulated in vari-
ous malignancies including melanoma . Ramirez et al.
 suggested higher level of cyclin D1 expression in pri-
mary melanoma compared with melanocytic nevus. Also,
Preferentially expressed Antigen in Melanoma (PRAME),
a tumor-associated antigen isolated by autologous T cells
in a melanoma patient, was reported to be overexpressed
in malignant melanomas rather than benign melanocytic
lesions . Lezcano et al.  found that over 88% of
non-spindle cell primary cutaneous melanoma showed
diffuse positivity for PRAME while 86% of melanocytic
nevus were negative for PRAME.
However, IHC staining for cyclin D1 and PRAME has
not been thoroughly investigated in melanocytic lesions of
nail unit. The prevalence of subungual melanoma in Asian
population is quite higher than Western population; sub-
ungual melanoma has been reported to comprise about
10–18% of cutaneous melanoma in Asian population [12,
13]. Relatively poor prognosis, distinct clinical features,
and diverse genetic mutation of subungual melanoma [14–
16], may indicate differences in the biology of subungual
melanoma compared with other cutaneous melanoma.
Herein, we assessed the ability of promising two
markers, cyclin D1 and PRAME, to distinguish subun-
gual MIS from benign SMP, both clinically presenting as
melanonychia, in a retrospective study.
This retrospective study, approved by the Institutional
Review Board (IRB) of Asan Medical Center (IRB No.
2020 −1102), included cases of benign SMP including
lentigo and nevi, and subungual MIS, confirmed by bi-
opsy at Asan Medical Center from January 2016 to De-
cember 2020. Cases of melanocytic activation without
proliferation and melanoma where dermal invasion was
identified were excluded.
Variables of interest
Demographic and clinical data were investigated through
reviewing electronic medical record (EMR) of Asan
Medical Center, including sex, age at diagnosis, age of
onset, duration of disease, location of the lesion, width
of melanonychia, color of melanonychia, and presence of
nail dystrophy or periungual pigmentation including
Hutchinson’s sign and pseudo-Hutchinson’s sign.
Histopathologic data including melanocyte density,
confluency, pagetoid melanocytosis, presence of inflam-
matory cell infiltration or melanophage, cytologic atypia
of melanocytes, and distribution of melanin pigment
were investigated through reviewing the slides by two
dermatopathologists (YJK, CJJ) with agreement. Melano-
cyte density was measured as the number of intraepithe-
lial melanocytes over 1 mm dermoepidermal junction of
the nail matrix. As more than 30 cells per millimeter
was reported to suggestive of melanoma in situ , we
analyzed the slides according to whether melanocyte
density was over 30 cells per millimeter or not. Cytologic
atypia defined as nuclear enlargement, hyperchromatism,
or prominent nucleoli was graded as follows: absence,
mild, moderate, and severe.
Skin tissues obtained for routine diagnostic pathologic
examinations were used for IHC studies of the anti-
CYCLIN D1 (1:100, Mouse monoclonal, clone SP4, cata-
log No.CELL MARQUE, CELL MARQUE, Rocklin,Cali-
fornia, USA), anti-PRAME (1:1000, Rabbit polyclonal,
catalog No.ab219650, ABCAM, Cambridge, UK). Forma-
lin fixed, paraffin-embedded tissue sections were immu-
nohistochemically stained for expression of anti-
CYCLIN D1 and PRAME using a BenchMark XT auto-
matic immunostaining device (Ventana Medical Systems,
Kim et al. Diagnostic Pathology (2022) 17:41 Page 2 of 10
Tucson, AZ, USA) with ultraView Universal AP Red De-
tection Kit (Ventana Medical Systems, Tucson, AZ,
USA) according to the manufacturer’s instructions.
Cyclin D1 and PRAME expression was assessed in
both benign SMP and subungual MIS by counting pro-
portion of melanocytes with nuclear positivity under
200x magnification, which was statistically analyzed to
determine cutoff value regarding sensitivities and speci-
ficities, using Youden index (specificity + sensitivity −1).
Chi-squared test and Fisher’s exact test were used to com-
pare the categorical variables of clinicopathologic features
of subungual benign melanocytic proliferation and subun-
gual melanoma in situ. Mann-Whitney U test was used
for the continuous variables of clinicopathologic features.
Sensitivities and specificities for PRAME were calculated
to determine cutoff value regarding discriminatory power
assessed by the area under the receiver operating charac-
teristic curve (AUC), using MedCalc (version 20.0, Med-
Calc Software Ltd, Ostend, Belgium). All analyses except
for were performed using SPSS (version 23.0, IBM Corp,
Armonk, NY). In this study, the p-value of ≤0.05 was con-
sidered statistically significant.
A total of 14 patients and 13 patients were diagnosed
with benign SMP and subungual MIS, respectively, at
Asan Medical Center from January 2016 to December
2020 (Supplementary Appendix 1). Of 14 cases of benign
SMP, 8 cases were diagnosed with subungual lentigo
while the others were subungual melanocytic nevus. The
demographic characteristics are summarized in Table 1.
The cohort of benign SMP included 7 males and 7 fe-
males with a mean age of 30.71 years (range, 7–66
years). The cohort of subungual MIS included 7 males
Table 1 Clinical features of subungual benign melanocytic proliferation and subungual melanoma in situ
Features Subungual benign melanocytic
proliferation (n= 14), n (%)
in situ (n= 13), n (%)
Male 7 (50) 7 (53.8)
Female 7 (50) 6 (46.2)
Age, years 0.061
Range 7-66 6-74
Mean ± SD 30.71 ± 19.83 44.92 ± 17.73
Onset age, years 0.048
Range 4-56 5-64
Mean ± SD 25.71 ± 15.97 39.46 ± 17.16
Prediagnosis duration, months 0.756
Range 8-240 6-180
Mean ± SD 59.21 ± 67.27 66.62 ± 64.12
Finger 12 (85.7) 9 (69.2)
Toe 2 (14.3) 4 (30.8)
Width, mm 0.017
Range 1.00-13.00 1.50-15.00
Mean ± SD 3.39 ± 2.90 7.81 ± 4.87
Background pigmentation 1.000
Yes 11 (78.6) 11 (84.6)
No 3 (21.4) 2 (15.4)
Nail dystrophy 0.596
Yes 0 (0.0) 2 (15.4)
No 14 (100.0) 11 (84.6)
Periungual pigmentation 0.568
Yes 6 (42.9) 7 (53.8)
No 8 (57.1) 6 (46.2)
Kim et al. Diagnostic Pathology (2022) 17:41 Page 3 of 10
and 6 females with a mean age of 44.92 years (range, 6–
74 years). The patients with subungual MIS seemed to
be older than the patients with benign SMP, but the dif-
ference was not statistically significant (p= 0.061).
Characteristics of subungual benign melanocytic
proliferation and subungual melanoma in situ
The clinical features of benign SMP and subungual MIS
are summarized in Table 1. The patients with benign
SMP were more likely to have melanonychia at younger
age (mean ± SD, 25.71 ± 15.9) than the patients with sub-
ungual MIS (mean ± SD, 39.46 ± 17.16) (p= 0.048). The
mean duration from the onset of the skin lesion to diag-
nosis of benign SMP was 59.21 months (range, 8-240
months); the mean prediagnosis duration of subungual
MIS was 66.62 months (range, 6-180 months). The fin-
gernail was involved in 12 patients with benign SMP
(85.7%) and 9 patients with subungual MIS (69.2%),
while toenail was involved in 2 patients with benign
SMP (14.3%) and 4 patients with subungual MIS
(30.8%). The width of melanonychia was significantly
thinner in benign SMP (mean ± SD, 3.39 ± 2.90 mm)
than subungual MIS (mean ± SD, 7.81 ± 4.87 mm) (p=
0.017). Colors of melanonychia striata were tan, brown,
or black, with background pigmentation in 11 out of 14
patients with benign SMP (78.6%) and 11 out of 13 pa-
tients with subungual MIS (84.6%). There was no patient
with benign SMP showing nail dystrophy at the involved
nail. Periungual pigmentation on proximal nail fold or
hyponychium suggestive of Hutchinson’s sign or
pseudo-Hutchinson’s sign was observed in 6 patients
with benign SMP (42.9%). When it comes to subungual
MIS, nail dystrophy and periungual pigmentation was
found in 2 patients (15.4%) and 7 patients (53.8%) re-
spectively, with no significant difference compared with
The histopathological features of benign SMP and sub-
ungual MIS are showed in Table 2. Melanocytic density
more than 30 cells per 1 mm stretch of subungual der-
moepidermal junction was not statistically different be-
tween subungual MIS and benign SMP (p= 0.182). Focal
confluency was identified significantly less in benign
SMP than subungual MIS (p< 0.001); 5 of 14 (35.7%)
cases showed focal confluency, while at least focal con-
fluency was found in all cases of subungual MIS. The
patients with benign SMP showed significantly less page-
toid melanocytosis (6 out of 14 patients) than subungual
MIS (12 out of 13) (p= 0.006). Inflammatory cell infiltra-
tion was found in 5 of 14 patients with benign SMP
(35.7%) and 5 of 13 patients with subungual MIS
(38.5%), respectively. Compared with subungual MIS, cy-
tologic atypia of benign SMP was significantly milder
(p< 0.001); 12 out of 14 patients with benign SMP
(85.7%) showed absent or mild cytologic atypia, while 10
out of 13 patients with subungual MIS (76.9%) showed
moderate to severe atypia. In benign SMP cases, mela-
nophage was found in 8 cases (57.1%), and melanin pig-
ment limited to basilar area was identified in 4 cases
(28.6%). In subungual MIS cases, melanophage was
found in 9 cases (69.2%), and melanin pigment in entire
epidermis was found in 9 cases (69.2%). More specific-
ally, out of 4 benign SMP showing melanocytic density >
30 cells/mm, 2 cases were lentigines and 2 cases were
nevi. All 5 benign SMP with confluency were nevi, 5 out
of the 6 benign SMP cases with pagetoid melanocytosis
Table 2 Histopathological features of subungual benign
melanocytic proliferation and subungual melanoma in situ
Features Subungual benign
(n= 14), n (%)
situ (n= 13),
Melanocyte density 0.182
≤30 cells/mm 10 (71.4) 6 (46.2)
> 30 cells/mm 4 (28.6) 7 (53.8)
Confluency < 0.001*
Yes 5 (35.7) 13 (100.0)
No 9 (64.3) 0 (0.0)
Pagetoid melanocytosis 0.006*
Yes 6 (42.9) 12 (92.3)
No 8 (57.1) 1 (7.7)
Yes 5 (35.7) 5 (38.5)
No 9 (64.3) 8 (61.5)
Atypia < 0.001*
No 6 (42.9) 0 (0.0)
Mild 6 (42.9) 3 (23.1)
Moderate 2 (14.3) 5 (38.5)
Severe 0 (0.0) 5 (38.5)
Yes 8 (57.1) 9 (69.2)
No 6 (42.9) 4 (30.8)
Melanin pigment 1.000
Basilar 4 (28.6) 4 (30.8)
Entire 10 (71.4) 9 (69.2)
cyclin D1 1.000
Positive 4 (28.6) 3 (23.1)
Negative 7 (71.4) 9 (66.9)
Positive 1 (7.1) 10 (76.9)
Negative 13 (92.9) 3 (23.1)
Abbreviations: PRAME PReferentially expressed Antigen in MElanoma
Kim et al. Diagnostic Pathology (2022) 17:41 Page 4 of 10
were lentigines, and 1 case was nevus. Also, out of 8 be-
nign SMP with atypia, 5 lentigines and 1 nevus showed
mild atypia, and 2 nevi showed moderate atypia.
The results of IHC staining are summarized in Table 3.
When it comes to benign SMP, four cases (28.6%)
showed ≤20% nuclear immunostaining for cyclin D1,
while 11 cases (71.4%) exhibited > 70% nuclear immuno-
staining for cyclin D1. 13 cases (92.9%) exhibited totally
negative staining for PRAME, while 1 case (7.1%)
showed ≤20% nuclear immunostaining for PRAME
(Fig. 1). In terms of subungual MIS, eight cases (61.5%)
showed ≤20% nuclear immunostaining for cyclin D1,
while 5 cases (38.5%) exhibited > 60% nuclear immuno-
staining for cyclin D1. 3 cases (23.1%) were totally nega-
tive for PRAME, while 10 cases (76.9%) exhibited > 50%
nuclear immunostaining for PRAME (Fig. 2).
Sensitivity and specificity of cyclin D1 and PRAME
Using the cutoff of 92.5%, with > 92.5% nuclear immu-
nostaining for cyclin D1 as a positive test for subungual
MIS, the sensitivity, specificity, positive predictive value
(PPV), and negative predictive value (NPV) of cyclin D1
to distinguish subungual MIS from benign SMP were
23.1%, 85.7%, 21.6%, and 91.7%, respectively. At this cut-
off, cyclin D1 showed poor overall discrimination be-
tween benign SMP and subungual MIS (AUC = 0.527,
95% CI = 0.328–0.721) (Fig. 3). There was no significant
differences in the positivity of cyclin D1 with cutoff of
92.5% between two groups (p= 1.000) (Table 2).
Using the cutoff of 10%, with > 10% nuclear immuno-
staining for PRAME as a positive test for subungual
MIS, the sensitivity, specificity, PPV, and NPV of
PRAME for distinguishing subungual MIS from benign
SMP were 76.9%, 92.9%, 71.6%, and 99.8%, respectively.
At this cutoff, PRAME exhibited good overall discrimin-
ation between benign SMP and subungual MIS (AUC =
0.849, 95% CI = 0.659–0.957) (Fig. 3), which distin-
guished two groups significantly better than cyclin D1
(p= 0.013). PRAME was significantly more expressed in
subungual MIS than benign SMP (p< 0.001) (Table 2).
Distinguishing subungual melanoma from non-nail ap-
paratus acral melanoma is clinically important due to its
distinct clinical courses including high recurrence rate
and short progression-free survival [14,15]. Also, it has
been reported that subungual melanoma harbors more
distinct genomic alterations including CARD11,ARID2,
ARID1A,ARID1B,PTPRB, and PTPRK genes, compared
with acral melanoma .
Cyclin D1 in coordination with their catalytic partners
CDK4 and CDK6, contributes to promoting cell cycle
progression through transition from G1 to S phase .
Cyclin D1 upregulation with amplification of CCND1
has been documented in various malignancies including
breast, lung, colon, and oral cancers . Meanwhile,
the expression rate of cyclin D1 in cutaneous melanoma
compared with benign melanocytic nevus has been dis-
cordantly reported [9,20,21]. When it comes to acral
melanoma, cyclin D1 has been reported to be overex-
pressed, resulting in constitutively activated MAPK sig-
naling pathway without NRAS or BRAF mutations [22–
24]. In this regard, we had expected cyclin D1 IHC stain-
ing would be an effective discriminator of benign SMP
from subungual MIS.
However, cyclin D1 IHC staining seemed not to be re-
liable in distinguishing benign SMP from subungual
MIS, in this study. Positive nuclear immunostaining for
cyclin D1 was found in 4 out of 14 (28.6%) patients with
benign SMP and 3 out of 13 (23.1%) patients with sub-
ungual MIS using cutoff of 92.5% which was determined
Table 3 Immunohistochemistry staining of subungual benign
melanocytic proliferation and subungual melanoma in situ
(n= 14), n (%)
(n= 13), n (%)
cyclin D1 nuclear
0% 2 (14.3) 2 (15.4)
>0–10% 0 (0.0) 2 (15.4)
>10–20% 2 (14.3) 4 (30.8)
>20–30% 0 (0.0) 0 (0.0)
>30–40% 0 (0.0) 0 (0.0)
>40–50% 0 (0.0) 0 (0.0)
>50–60% 0 (0.0) 0 (0.0)
>60–70% 0 (0.0) 1 (7.7)
>70–80% 3 (21.4) 0 (0.0)
>80–90% 3 (21.4) 1 (7.7)
>90–100% 4 (28.6) 3 (23.1)
0% 13 (92.9) 3 (23.1)
>0–10% 0 (0.0) 0 (0.0)
>10–20% 1 (7.1) 0 (0.0)
>20–30% 0 (0.0) 0 (0.0)
>30–40% 0 (0.0) 0 (0.0)
>40–50% 0 (0.0) 0 (0.0)
>50–60% 0 (0.0) 3 (23.1)
>60–70% 0 (0.0) 0 (0.0)
>70–80% 0 (0.0) 1 (7.7)
>80–90% 0 (0.0) 2 (15.4)
>90–100% 0 (0.0) 4 (30.8)
Abbreviations: PRAME PReferentially expressed Antigen in MElanoma
Kim et al. Diagnostic Pathology (2022) 17:41 Page 5 of 10
regarding sensitivities and specificities using Youden
index, with poor sensitivity of 23.1%. The lack of diag-
nostic value of cyclin D1 overexpression in these lesions
may be due to the differences in the type of antibody
used, the positive cell count system, and cutoff point for
positivity . Also, it is important to note that cyclin D1
overexpression does not always represent amplification
of CCND1; loss of cyclin D1 IHC staining does not al-
ways represent loss of function of CCND1, vice versa.
Epigenetic regulation including DNA methylation at
cytosine and histone acetylation can cause alteration in
mRNA and protein expression . Therefore, further
genetic tests including fluorescent in situ hybridization
(FISH) for CCND1 may be needed for determining true
CCND1 amplification to discriminate between benign
SMP and subungual MIS .
PRAME gene is a member of cancer testis antigen
(CTA) gene family encoding a membrane-bound protein
recognized by T lymphocytes, causing autologous cyto-
toxic T cell-mediated immune response . Except for
some distinct tissues including testis, ovary, placenta, ad-
renals, and endometrium, PRAME is not detected in
healthy human tissues . Overexpression of PRAME
was found to inhibit retinoic acid (RA) mediated cell dif-
ferentiation, cell growth arrest, and apoptosis, contribut-
ing to tumorigenesis via inhibiting RA receptor signaling
. PRAME has been reported to be overexpressed in a
variety of malignancies including malignant melanoma,
showing an utility in distinguishing between benign and
malignant lesions . Although little has been studied
about the diagnostic utility of PRAME in subungual mel-
anocytic lesions, a recently published paper has validated
the usefulness of PRAME expression in differentiating
between melanoma and other nail unit melanocytic le-
sions . In the paper, subungual melanomas were all
positive in PRAME IHC, and benign melanocytic lesions
of nail unit were all negative. However, out of the 25
melanoma cases, 20 cases were invasive melanoma,
while only 5 cases were MIS. Our paper provides add-
itional insight from current literature focusing on MIS,
which is more difficult to distinguish from benign
In this study, we demonstrated that PRAME IHC
staining was a relia4ble discriminator of benign SMP
from subungual MIS. Positive nuclear immunostaining
for PRAME was found in 1 out of 14 (7.1%) patients
Fig. 1 Representative clinical morphology, photomicrographs of H&E staining, and IHC staining for cyclin D1and PRAME of selected subungual
benign melanocytic proliferation. One 7-year-old patient presenting with (A) 3.5 mm-wide melanonychia showed (B) melanocyte proliferation
showing mild atypia without confluency or pagetoid spread (200x magnification, H&E). (C) While cyclin D1 IHC showed over 70% nuclear
immunostaining (200x magnification), (D) PRAME IHC exhibited total negativity (200x magnification), (E) Sox-10 IHC (200x magnification)
Kim et al. Diagnostic Pathology (2022) 17:41 Page 6 of 10
with benign SMP and 10 out of 13 (76.9%) patients with
subungual MIS using cutoff of 10%, showing modest
sensitivity of 76.9% and good specificity of 92.9%. Cutoff
(10%) for positivity of PRAME in this study is quite dif-
ferent from previously reported cutoff values ranging
from 50 to 75% [7,10,28]. Differences in cutoff values
among studies may be due to divergences in IHC stain-
ing methodology and inter-observer variability in IHC
assessment . Also, the type of melanocytic lesions in-
cluded in the studies might affect the differences in cut-
off value for positivity of PRAME. Unlike previous
studies, we assessed the expression of PRAME using
only subungual melanocytic lesions, known to have dif-
ferent genomic alterations with lower mutation burden
compared with other cutaneous melanomas [23,30].
Also, small sample size of this study might have influ-
enced the difference in cutoff value. Therefore, add-
itional studies of PRAME IHC in a large cohort of
melanocytic lesions with different subtypes are needed
to determine whether cutoff value for PRAME positivity
should differ according to the subtype of melanocytic le-
sions, or not.
Compared with cytogenetic studies including FISH,
IHC has several advantages including more rapid turn-
around time, lower cost, and higher accessibility. How-
ever, PRAME IHC can exhibit false positive and false
negative results, confusing the correct diagnosis. Lez-
cano et al.  reported that diffuse nuclear immunore-
activity for PRAME was found in 13.6% of cutaneous
melanocytic nevus. Also, Shyam et al.  demonstrated
focal immunopositivity of PRAME from 5 to 10% in
atypical but benign non-spitzoid melanocytic prolifera-
tions. In this study, 1 out of 14 (7.1%) patients with be-
nign SMP exhibited PRAME nuclear immunostaining
ranging from 10 to 20%. The significance of focal
PRAME positivity in benign melanocytic lesions is still
Fig. 2 Representative clinical morphology, photomicrographs of H&E staining, and IHC staining for cyclin D1 and PRAME of selected subunugal
melanoma in situ. One 44-year-old patient (A) 2 mm-wide melanonychia showed (B) atypical melanocyte proliferation with confluency and
pagetoid spread (200x magnification, H&E). Both (C) cyclin D1 and (D) PRAME IHC showed over 90% nuclear immunostaining (200x magnification,
respectively), (E) Sox-10 IHC (200x magnification)
Kim et al. Diagnostic Pathology (2022) 17:41 Page 7 of 10
unclear, which necessitate further studies to determine
the potential risk of malignant transformation related to
focal PRAME positivity. Additional cytogenetic and mo-
lecular tests can help avoiding overdiagnosis in the cases
with focal PRAME positivity .
Also, it has been reported that primary cutaneous
melanoma could be completely negative for PRAME
immunostaining . In this study, 3 out of 13 (23.1%)
patients showed completely negative PRAME IHC.
Histopathological analysis revealed there was no mela-
nophage adjacent to cutaneous melanoma in these pa-
tients. Melanophage found in cutaneous melanoma is
indicative of immune responses, predicting a relatively
good prognosis for patients, possibly through tumor re-
gression due to phagocytosis of melanoma cells . As
PRAME gene encodes a membrane-bound protein rec-
ognized by T lymphocytes causing autologous cytotoxic
T cell-mediated immune response , it is possible
that lack of PRAME expression may lead to decreased
immune response and reduced melanophages. While
PRAME was found to be related with metastasis in
uveal melanoma , little has been reported about the
prognostic value of PRAME expression in cutaneous
melanoma. Further studies are needed to determine the
prognostic value of PRAME expression in cutaneous
melanoma, regarding the role of PRAME in the rela-
tionship between immune response and oncogenesis.
It is important to keep in mind that IHC should be
used in conjunction with clinical findings and histo-
logical analysis to make a final diagnosis. Clinically, we
found that the patients with subungual MIS tended to
be older and have wider lesions than the patients with
benign SMP, with statistical significance. We also dem-
onstrated that the patients with subungual MIS showed
significantly more confluency, pagetoid melanocytosis,
and severe atypia than the patients with benign SMP.
Combining these clinical and histopathological features
with PRAME IHC may significantly increase diagnostic
power to distinguish benign SMP from subungual MIS.
This study has several limitations. First, this study was
a retrospective study with relatively small sample size.
Small sample size limited the usage of multivariate logis-
tic regression analysis to develop a scoring system for
the diagnosis of subungual melanoma in situ using
PRAME expression. Second, this study was a single-
center study confined to Korean patients. As the differ-
ence in the prevalence rate of subungual melanoma be-
tween the Asian group and other races including
Fig. 3 Receiver Operating Characteristic (ROC) curve for discriminating subungual MIS from benign SMP based on the expression levels of Cyclin
D1 and PRAME. Using the cutoff of 92.5%, with > 92.5% nuclear immunostaining for cyclin D1 as a positive test for subungual MIS, cyclin D1
showed poor overall discrimination between benign SMP and subungual MIS (AUC = 0.527, 95% CI = 0.328–0.721). Using the cutoff of 10%, with
> 10% nuclear immunostaining for PRAME as a positive test for subungual MIS, PRAME exhibited good overall discrimination between benign
SMP and subungual MIS (AUC = 0.849, 95% CI = 0.659–0.957)
Kim et al. Diagnostic Pathology (2022) 17:41 Page 8 of 10
Western group is prominent enough to suppose differ-
ence in the biology of subungual melanoma in each
group. Therefore, cyclin D1 and PRAME expression in
melanocytic lesions of nail unit should be studied in
other races including Western group. Also, this study
evaluated no cytogenetic or molecular studies to com-
pare with PRAME IHC in the diagnosis of subungual
This study suggests that PRAME IHC staining as a reli-
able discriminator in distinguishing subungual MIS from
benign SMP. We also demonstrated PRAME expression
should be interpreted in the context of histopathologic
features. Further studies using larger cohort and ancil-
lary studies including cytogenetic studies are needed to
confirm the diagnostic utility of cyclin D1 and PRAME
expression in the diagnosis of subungual MIS.
IHC: Immunohistochemistry; MIS: Melanoma in situ; SMP: Subungual
melanocytic proliferation; PRAME: Preferentially expressed Antigen in
Melanoma; PPV: Positive predictive value; NPV: Negative predictive value;
CTA: Cancer testis antigen; RA: Retinoic acid
The online version contains supplementary material available at https://doi.
Additional file 1: Supplementary Appendix 1. Clinical and
histopathological data for all cases included.
We would like to thank all patients who participated in the study. We would
like to thank the National Research Foundation of Korea for providing
YJK and CJJ designed the study, collected the clinical, histopathological data,
analyzed the data, and wrote the manuscript. CSP performed
immunohistochemical study. All authors commented on previous versions of
the manuscript. All authors read and approved the final manuscript.
This research was supported by the National Research Foundation of Korea
(NRF) grant funded by the Korean government (MSIT). (No.
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Ethics approval and consent to participate
This retrospective study was approved by the Institutional Review Board (IRB)
of Asan Medical Center (IRB No. 2020 −1102).
Consent for publication
All the patients involved in this study gave their written informed consent
for publication of their case details.
The authors declare that they have no competing interests.
Department of Dermatology, Asan Medical Center, University of Ulsan
College of Medicine, 88 Olympic- ro 43 gil, Songpa-gu, Seoul, Korea.
Department of Pathology, Asan Medical Center, University of Ulsan College
of Medicine, 88 Olympic- ro 43 gil, Songpa-gu, Seoul, Korea.
address: Department of Dermatology, Seoul National University Hospital, 101,
Daehak ro, Jongno gu, Seoul, Korea.
Received: 9 November 2021 Accepted: 5 April 2022
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