Journal of Cancer Research and Clinical Oncology (2018) 144:257–267

https://doi.org/10.1007/s00432-017-2550-z

ORIGINAL ARTICLE – CANCER RESEARCH

Analysis of TSC1 mutation spectrum in mucosal melanoma

Meng Ma1 · Jie Dai1 · Tianxiao Xu1 · Sifan Yu1 · Huan Yu1 · Huan Tang1 · Junya Yan1 · Xiaowen Wu1 · Jiayi Yu1 ·
Zhihong Chi1 · Lu Si1 · Chuanliang Cui1 · Xinan Sheng1 · Yan Kong1 · Jun Guo1

Received: 1 August 2017 / Accepted: 23 November 2017 / Published online: 28 November 2017
© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract
Purpose Mucosal melanoma is a relatively rare subtype of melanoma for which no clearly established therapeutic strategy
exists. The genes of the mTOR signalling pathway have drawn great attention as key targets for cancer treatment, including
melanoma. In this study, we aimed to investigate the mutation status of the upstream mTOR regulator TSC1 and evaluated
its correlation with the clinicopathological features of mucosal melanoma.
Methods We collected 91 mucosal melanoma samples for detecting TSC1 mutations. All the coding exons of TSC1 were
amplified by PCR and subjected to Sanger sequencing. Expression level of TSC1 encoding protein (hamartin) was detected
by immunohistochemistry. The activation of mTOR pathway was determined by evaluating the phosphorylation status of
S6RP and 4E-BP1.
Results The overall mutation frequency of TSC1 was found to be 17.6% (16/91 patients). TSC1 mutations were more inclined
to occur in advanced mucosal melanoma (stages III and IV). In the 16 patients with TSC1 mutations, 14 different mutations
were detected, affecting 11 different exons. TSC1 mutations were correlated with upregulation of S6RP phosphorylation but
were unrelated to 4E-BP1 phosphorylation or hamartin expression. Mucosal melanoma patients with TSC1 mutations had
a worse outcome than patients without TSC1 mutations (24.0 versus 34.0 months, P = 0.007).
Conclusions Our findings suggest that TSC1 mutations are frequent in mucosal melanoma. TSC1 mutations can activate the
mTOR pathway through phospho-S6RP and might be a poor prognostic predictor of mucosal melanoma. Our data implicate
the potential significance of TSC1 mutations for effective and specific drug therapy for mucosal melanoma.

Keywords TSC1 · Hamartin · Mucosal melanoma · Mutation · mTOR pathway

Introduction cutaneous melanoma, mucosal melanoma is a relatively

rare subtype of melanoma. The incidence rate of mucosal
Mucosal melanoma originates from melanocytes in mucosal melanoma in the Asian population appears to differ from
membranes lining respiratory, gastrointestinal, and geni- that among Caucasians. Mucosal melanoma comprises
tourinary tracts (Mihajlovic et al. 2012). Compared with approximately 1.3% of all melanocytic malignancies in
America (Chang et al. 1998), whereas it constitutes 24% of
all melanomas in China (Chi et al. 2011). Mucosal mela-
Electronic supplementary material The online version of this noma is poorly characterised and incompletely understood
article (https://doi.org/10.1007/s00432-017-2550-z) contains owing to its rare occurrence in most countries. Mucosal
supplementary material, which is available to authorized users. melanoma has been found to have distinct biological and
* Yan Kong clinical features (Chi et al. 2011; Curtin et al. 2005), lower
k‑yan08@163.com somatic mutational burden (Furney et al. 2013; Hayward
* Jun Guo
et al. 2017; Kong et al. 2011), later diagnosis, and signifi-
guoj307@126.com cantly worse outcomes (Chang et al. 1998; Kuk et al. 2016)
compared with cutaneous melanomas. A whole-genome
1
Key Laboratory of Carcinogenesis and Translational sequencing analysis of different melanoma subtypes mani-
Research (Ministry of Education/Beijing), Department
of Renal Cancer and Melanoma, Peking University Cancer
fested that mucosal melanoma showed a significantly dif-
Hospital and Institute, 52 Fucheng Road, Beijing 100142, ferent genomic landscape from cutaneous melanoma, with
China

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258 Journal of Cancer Research and Clinical Oncology (2018) 144:257–267

large-scale structural variants leading to a far lower muta- may be useful for selection of mucosal melanoma patients
tion burden. Meanwhile, mutation frequencies of previ- who may benefit from mTOR pathway inhibitors.
ously reported melanoma driver genes varied considerably To explore potential targets for mucosal melanoma ther-
between mucosal and skin melanomas, indicating that the apy, we evaluated the genetic mutations and protein expres-
molecular pathways driving mucosal melanoma differ mark- sion of TSC1 in mucosal melanoma and further analysed the
edly from those driving cutaneous melanoma (Hayward activation of the mTOR pathway by detecting phosphoryla-
et al. 2017). Targeted therapies, such as BRAF inhibitors tion level of 4E-BP1 and S6RP. Our study might provide
and CKIT inhibitors, as well as immunotherapies, have revo- potential targets for mucosal melanoma treatment or clues
lutionised the treatment of cutaneous melanoma. However, about application of mTOR pathway inhibitors in mucosal
the effect of such targeted therapy and immunotherapy on melanoma, as well as new insights into the molecular biol-
the treatment for mucosal melanoma was unclear due to the ogy of mucosal melanoma.
limited numbers of mucosal melanoma patients included in
most clinical trials. Besides, the mutation rates of the BRAF
and CKIT genes were only about 13.9 and 9.6%, respec- Materials and methods
tively, in mucosal melanoma patients (Kong et al. 2011, Si
et al. 2012). Thus, validated targets should be identified for Patients and samples
mucosal melanoma patients for developing new and effective
molecularly targeted therapies. A total of 91 mucosal melanoma patients that were hos-
TSC1 and TSC2 (tuberous sclerosis complex 1 and 2) pitalised during January 2007–December 2013 at Peking
are two tumour-suppressor genes responsible for an auto- University Cancer Hospital and Institute were involved in
somal dominant disorder, tuberous sclerosis complex. The this study. The diagnosis of melanoma of these samples was
TSC1 and TSC2 gene products, hamartin and tuberin, form confirmed by haematoxylin and eosin staining and immuno-
a physical and functional complex with TBC1D7 (Tre2- histochemistry (IHC) for melanoma markers (S-100, HMB-
Bub2-Cdc16-1 domain family member 7), known as the TSC 45, or MART-1). Clinical data were collected, including sex,
protein complex (Plank et al. 1998; Tee et al. 2002, 2003). age, tumour–node–metastases (TNM) stage, thickness (Bres-
TSC1 is located on chromosome 9q34 (MIM 191100), which low thickness), ulceration, and overall survival (OS; until
contains 21 coding and 2 leader exons and encodes an 8.6 kb loss to follow-up or death of patients). Pathological data
mRNA (van Slegtenhorst et al. 1997). TSC1 is important for such as mucosal origin, TNM stage, ulceration and thickness
maintaining the stability, activity, and correct intracellular were identified by two pathologists separately.
localisation of the TSC complex (Benvenuto et al. 2000;
Chong-Kopera et al. 2006). The TSC protein complex exerts DNA extraction and sequencing
GTPase-activating protein (GAP) activity towards a small
G protein, Rheb, activating mammalian target of rapamycin Genomic DNA was extracted from formalin-fixed, paraf-
complex 1 (mTORC1) (Tee et al. 2003). mTORC1 is located fin-embedded (FFPE) specimens using the QIAamp DNA
deep within signalling pathway cascades, downstream of FFPE tissue kits (Qiagen, Hilden, Germany) according to the
both PI3K and MAPK signalling pathways (Zhang et al. manufacturer’s instructions. Exons 3 to 23 of the TSC1 gene
2014) regulating anabolic metabolism, including protein were amplified by PCR. The primer sequences are listed in
synthesis, ribosomal biogenesis, lipogenesis, and mitochon- Online Resource 1.
drial biogenesis (Dodd and Dunlop 2016; Zhang et al. 2014).
Eukaryotic initiation factor 4E (eIF4E)-binding protein-1 Prediction of functional mutations
(4E-BP1) and p70 ribosomal protein S6 kinase 1 [p70S6K,
which in turn phosphorylate the 40S ribosomal protein S6 MutationAssessor (http://mutationassessor.org/r3/), Poly-
(S6RP)] are two proteins whose phosphorylation starts the Phen (http://genetics.bwh.harvard.edu/pph/) and SIFT
translation process (Janus et al. 2005). The mTOR pathway (http://sift.jcvi.org/) were used for amino acid substitution
has been found to play a pivotal role in melanoma genesis functional prediction. Protein sequence, NP_000359.1, was
and progression (Dai et al. 2005; Meier et al. 2005), but used if needed.
TSC1 mutations have never been comprehensively inves-
tigated in melanoma. In an earlier study, we found that Immunohistochemistry
patients with specific mTOR mutations could benefit from
PI3K–AKT–mTOR pathway inhibitors (Kong et al. 2016), Antibodies against hamartin (1:100, Abcam, Cambridge,
but the rate of mTOR gene mutation was only 14.3% in UK), phospho-S6RP (Ser235/236, 1:400, Cell Signaling
mucosal melanoma. Inactive mutations of TSC1 might in Technology, Danvers, CA), and phospho-4E-BP1 (Thr37/46,
turn upregulate mTOR activity, detection of TSC1 mutations 1:1000, Cell Signaling Technology, Danvers, CA) were used

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Journal of Cancer Research and Clinical Oncology (2018) 144:257–267 259

for IHC analysis. Dako polyclonal rabbit antibody (DakoCy- Table 1  Genetic mutations of TSC1 in mucosal melanoma
tomation, Glostrup, Denmark) was used at a 1:400 dilution Anatomic site Number of cases Number of cases
followed by visualisation with 3-amino-9-ethylcarbazole with mutation (%)
(Solarbio, Beijing, China) and counterstaining with haema-
Head and n­ ecka 43 3 (7.0)
toxylin. The staining intensity was scored as “0” (negative),
Genitourinaryb 22 6 (27.3)
“1” (weak), “2” (moderate), and “3” (strongest). Examples
Anorectum 20 5 (25.0)
of the scores are shown in Online Resource 2.
Oesophagus 6 2 (33.3)
Total 91 16 (17.6)
Statistical analysis
a
The sites of head and neck mucosal melanomas included nasal cav-
Statistical analyses were performed using SPSS 20.0 soft- ity, paranasal sinuses and oral cavity
ware (IBM Corp., Armonk, NY, USA). Continuous data b
The sites of genitourinary mucosal melanomas included vulva,
such as age and Breslow thickness were described using vagina, urethra and cervix
means ± SD for normally distributed data. Categorical data
were described using frequencies and percentages. The cor- anatomic sites of mucosal melanoma (P = 0.088). However,
relations between TSC1 mutation or hamartin expression sta- when taken mucosal melanoma arising from genitourinary,
tus and clinical parameters were evaluated by the Pearson’s anorectum, and oesophagus together, the mutation rate in
Chi-square test. Survival curves were established using the the head and neck (7.0%) was significantly lower than that
Kaplan–Meier method and compared by the log-rank test. in other types (27.1%, P = 0.012).
All statistical analyses were two sided, and significance Among the 16 cases with TSC1 mutations, 14 different
was assigned at P < 0.05. GraphPad Prism 6 (GraphPad nonsynonymous mutations were detected, including 12
Software, LaJolla, CA, USA) was used for calculation and heterozygous point mutations, 1 homozygous point muta-
graphs’ design. tion, and 1 inframe deletion mutation. TSC1 nonsynony-
mous mutations did not cluster defined hotspots. However,
2 recurrent TSC1 mutations were found: M322T (2 cases)
Results and Y761H (2 cases). Nonsynonymous TSC1 mutations
were detected in 11 of the 21 exons, which affected the
Baseline patient characteristics Rho-activating domain (amino acids 145–510, 5 cases),
tuberin-binding domain (amino acids 302–430, 3 cases),
A total of 91 mucosal melanoma patients were screened for and coiled-coil region (amino acids 730–996, 6 cases)
TSC1 mutations. These patients are divided into four types (Fig. 1). Furthermore, 10 different synonymous mutations
according to the anatomic sites of mucosal melanoma, the in 24 patients were found. The most common synonymous
head and neck (nasal pharyngeal and oral, 47.2%, 43/91), mutations were of codon 445 in exon 14 (9/24 patients),
genitourinary tract (vulva, vagina, urethra and cervix, 24.2%, followed by codon 943 in exon 22 (5/24) and codon 576 in
22/91), anorectum (22.0%, 20/91), and oesophagus (6.6%, exon 15 (3/24) (Fig. 1). Hereafter, “TSC1 mutation” refers
6/91). The proportion of patients with TNM stage I, II, III, to a nonsynonymous mutation in TSC1.
and IV disease was 1.1, 40.6, 20.9, and 37.4%, respectively. To assess if these mutations are pathogenic, we used three
Thickness data of 55 samples and ulceration status of 82 well-known methods: SIFT (Kumar et al. 2009), PolyPhen2
patients were available: 57.3% patients (47/82) had ulcera- (Adzhubei et al. 2010) and MutationAssessor (Reva et al.
tion and 42.7% patients did not (35/82). 2011). The results indicated that four changes were prob-
able to be deleterious (S108P, S108F, L459V, Q701R) as
Genetic aberrations of TSC1 gene in mucosal they were predicted to affect protein function by all the three
melanoma methods. According to MutationAssessor and PolyPhen, two
changes (P196H, R922G) were predicted to have possible
Owing to the broad spectrum of TSC1 gene mutations, we impact on hamartin function, while they are predicted to
amplified the 21 coding exons (exons 3 to 23) of the TSC1 be tolerated in SITF. And three mutations (Y761H, F847L,
gene for Sanger sequencing. The overall frequency of TSC1 S840T) were predicted to be probably damaging by Poly-
mutations was found to be 17.6% (16/91 patients). The muta- Phen, have low impact on hamartin function by Mutation-
tion frequencies of the TSC1 gene in mucosal melanoma Assessor but be tolerated by SITF. These are summarized
arising from the head and neck, genitourinary tract, anorec- in Online Resource 3.
tum, and oesophagus were 7.0% (3/43), 27.3% (6/22), 25.0% To exclude the possibility that the detected mutations
(5/20), and 33.3% (2/6), respectively (Table 1). There was no were due to polymorphisms, we extracted DNA from the
significant difference for TSC1 mutations among the primary peripheral blood lymphocytes of the 16 patients harbouring

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260 Journal of Cancer Research and Clinical Oncology (2018) 144:257–267

Fig. 1  Distribution of TSC1 mutations according to TSC1 domains. Amino acid positions of reported functional domains of hamartin are
TMD transmembrane domain (127–144), RAD rho-activating domain shown. The solid lines indicate for nonsynonymous mutations, with
(145–510), TBD tuberin-binding domain (302–430), CCR coiled- the dotted lines for synonymous mutations. The repetitive mutations
coil region (730–996), ERMD ERM interaction domain (881–1084). are indicated with “asterisk”

the 14 mutations and examined the mutation status of TSC1 (exon 15), and NRAS (exons 1 and 2), in the patients. The
in the corresponding exon. No mutations were detected, mutation rates of CKIT, BRAF, and NRAS in our cohort were
indicating that the mutations detected were indeed somatic 8.6% (7/81), 20% (13/65), 12.5% (8/64), separately. Most
mutations. patients with TSC1 mutations simultaneously harboured
To analyse the relationship of TSC1 mutations to mutations in one or more of these three genes while only
the mutation status of its downstream mTOR gene, we five patients bearing TSC1 mutations did not contain CKIT,
screened the whole 58 exon sequence of mTOR as previ- BRAF, or NRAS mutations (Table 2). Thus, it appears that
ously described (Kong et al. 2016). A total of 14 different TSC1 mutations might not be mutually exclusive to the other
mutations were found in 12 patients. In the 16 cases with genetic mutations of the previously validated targets. Statis-
TSC1 mutations, only 1 case was found to harbour mTOR tical analysis showed that there was no significant difference
(S616F) and TSC1 (Y761H) gene mutations simultaneously in the mutation frequency of these genes between patients
(Table 2). with or without TSC1 mutations. Therefore, TSC1 mutations
We also analysed the mutation status of important mela- might be unrelated to the other mutations such as CKIT,
noma driver, including CKIT (exons 9, 11, 13, 17, 18), BRAF BRAF, and NRAS mutation.

Table 2  Mutation types of TSC1 mTOR CKIT BRAF NRAS Anatomic site
TSC1 and the relationship
with other genes in mucosal Exon Nucleotide Amino acid No.
melanoma
5 G250A A84T 1 WT WT V600E Y4C Anorectum
T322C S108P 1 WT WT WT WT Anorectum
C323T S108F 1 WT WT WT WT Genitourinary
7 C587A P196H 1 WT WT K601E WT Head and neck
10 T965C M322T 2 WT WT WT WT Genitourinary
WT WT V600E WT Oesophagus
13 A1273G M425V 1 WT WT WT WT Oesophagus
14 C1375G L459V 1 WT WT WT WT Genitourinary
15 C1598A P533H 1 WT N822Y WT WT Anorectum
17 A2102G Q701R 1 WT V560D WT WT Genitourinary
18 T2281C Y761H 2 WT WT WT WT Anorectum
S616F WT WT WT Genitourinary
19 A2446-2448 del AAG K816del 1 WT I789T WT WT Head and neck
20 C2541A F847L 1 WT WT V600K WT Head and neck
T2518A S840T 1 WT WT S616F WT Anorectum
22 A2974G R992G 1 WT WT WT G12S Genitourinary

WT wild type

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Journal of Cancer Research and Clinical Oncology (2018) 144:257–267 261

Detection of hamartin expression levels P = 0.309, respectively) (Tables 5, 6). For the cases with
and activation of the mTOR pathway in mucosal hamartin expression, the phosphorylation rate of S6RP
melanoma in harmatin expression-negative cases was 51.3% (20 of
39), which was significantly higher than that in cases with
All the 91 melanoma samples were subjected to IHC stain- hamartin positive (16 of 52, 30.8%; P = 0.048). However,
ing for analysing hamartin expression. Expression of hamar- the positive rate of phospho-4E-BP1 was not significantly
tin protein was observed in 58.1% (25/43) of melanoma from different between mucosal melanoma with hamartin loss
the head and neck, 59.1% (13/22) of melanoma from the and detection (P = 0.157), data are shown in Tables 5 and 6.
genitourinary tract, 50.0% (10/20) of melanoma from the Overall, these results indicate that both TSC1 mutations and
anorectum, and 66.7% (4/6) of melanoma from the oesopha- hamartin loss would activate the mTOR pathway through
gus (Table 3). The expression status of hamartin was not p70S6K–S6RP.
significantly different among melanomas from different pri-
mary anatomic sites (P = 0.875). Then, we analysed the cor- Correlation of TSC1 mutations and hamartin
relation of TSC1 mutation with hamartin expression. Among expression with the clinical features of mucosal
the 16 cases with TSC1 mutations, the IHC detection rate melanoma
for hamartin was 43.8% (7/16), which was not significantly
different from that in cases without TSC1 mutations (60.0%, We analysed the correlation of TSC1 mutations with the
45/75; P = 0.233), as shown in Table 4. These data indicate characteristics of patients, including sex, age, ulceration,
that TSC1 mutations might not necessarily lead to decreased
hamartin expression.
Table 4  Correlation of TSC1 genetic mutations to hamartin expres-
Given that the hamartin/tuberin complex can inhibit the sion
nutrient-mediated or growth factor-stimulated phosphoryla-
tion of 4E-BP1 and S6RP by negatively regulating mTORC1 Hamartin TSC1 mutation
signalling (Janus et al. 2005), we examined the activation Mutation WT
of these downstream molecules by IHC. The overall posi- a
IHC ­scores
tive rates of phospho-S6RP and phospho-4E-BP1 were 39.6
0 9 30
and 33.0%, respectively (Table 3). We found that the posi-
1 5 25
tive rate of phospho-S6RP in mucosal melanoma bearing
2 1 15
TSC1 mutations (62.5%, 10/16) was significantly higher than
3 1 5
those without TSC1 mutations (34.7%, 26/75; P = 0.039)
Positive/totalb 7/16 45/75
(Table 5). The positive rate of phospho-4E-BP1 in mucosal
Positive rate (%) 43.8 60.0
melanomas bearing TSC1 mutations (50.0%, 8/16) seems
P ­valuec 0.233
to be higher than those without TSC1 mutations (29.3%,
a
22/75) but the difference was not statistically significant The signal intensity of immunohistochemistry results were deter-
(P = 0.110, Table 6). Considering that mTOR mutations mined by three individual pathologists and scored as 0, 1, 2, and 3,
with score “0” as negative and score “3” as the strongest
could also activate the S6RP or 4E-BP1, we analysed the b
Samples with signal intensity of score 0 was regarded as hamartin
relationship between phospho-S6RP or phospho-4E-BP1 negative, scores 1, 2 or 3 were regarded as hamartin positive
expression and mTOR mutation simultaneously. Neither c
Positive rate of hamartin in cases with TSC1 mutation versus that in
the expression of phospho-S6RP nor phospho-4E-BP1 cases without TSC1 mutation, significance evaluated by Chi-square
was correlated with mTOR mutation status (P = 0.081 and tests

Table 3  Hamartin, phospho- Anatomic site Hamartin Phospho-S6RP Phospho-4E-BP1

S6RP and phospho-4E-BP1
expression in mucosal No. Positivea % No. Positivea % No. Positivea %
melanoma
Head and neck 43 18 41.9 43 14 32.6 43 12 27.9
Genitourinary 22 9 40.9 22 10 45.5 22 8 36.4
Anorectum 20 10 50.0 20 10 50.0 20 8 40.0
Oesophagus 6 2 33.3 6 2 33.3 6 2 33.3
Total 91 39 42.9 91 36 39.6 91 30 33.0
a
The signal intensity of immunohistochemistry results were determined by three individual pathologists
and scored as 0, 1, 2, and 3, with score “0” as negative and score “3” as the strongest. Samples with signal
intensity of scores 1, 2 or 3 were regarded as positive

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Table 5  Correlation of Phospho-S6RP TSC1 mutation mTOR mutation Hamartin expression

phospho-S6RP expression to
TSC1 mutation, mTOR mutation Mutation WT Mutation WT Negative Positiveb
and hamartin expression
IHC ­scoresa
0 6 49 4 51 19 36
1 7 15 5 18 13 9
2 2 6 1 6 3 5
3 1 5 2 4 4 2
Positive/total 10/16 26/75 8/12 28/79 20/39 16/52
Positive rate (%)b 62.5 34.7 66.7 35.4 51.3 30.8
P ­valuec 0.039 0.081 0.048
a
The signal intensity of immunohistochemistry results were determined by three individual pathologists
and scored as 0, 1, 2, and 3, with score “0” as negative and score “3” as the strongest
b
Samples with signal intensity of scores 1, 2 or 3 were regarded as positive
c
Positive rate in cases with gene mutation versus that in cases without gene mutation, and in cases with
hamartin loss versus expression, significance evaluated by Chi-square tests

Table 6  Correlation of Phospho-4E-BP1 TSC1 mutation mTOR mutation Hamartin expression

phospho-4E-BP1 expression to
TSC1 mutation, mTOR mutation Mutation WT Mutation WT Negative Positiveb
and hamartin expression
IHC ­scoresa
0 8 53 6 55 23 38
1 4 13 3 14 9 8
2 2 6 2 6 4 4
3 2 3 1 4 3 2
Positive/total 8/16 22/75 6/12 24/79 16/39 14/52
Positive rate (%)b 50.0 29.3 50.0 30.4 41.0 26.9
P ­valuec 0.110 0.309 0.157
a
The signal intensity of immunohistochemistry results were determined by three individual pathologists
and scored as 0, 1, 2, and 3, with score “0” as negative and score “3” as the strongest
b
Samples with signal intensity of scores 1, 2 or 3 were regarded as positive
c
Positive rate in cases with gene mutation versus that in cases without gene mutation, and in cases with
hamartin loss versus expression, significance evaluated by Chi-square tests

tumour thickness, clinical stage, and primary anatomic sites, The average thickness of samples harbouring TSC1
as summarised in Table 7. There were no significant differ- mutations was 6.27 ± 3.55 mm, whereas that of samples
ences for sex and median age of mucosal melanoma patients with wild-type TSC1 was 5.19 ± 4.17 mm, which was
with or without TSC1 mutations. The proportions for sex and not significantly different (P = 0.434) (Table 7). Moreo-
median age were also similar between groups with hamartin ver, the average thickness of samples with hamartin loss
loss or without. was 6.02 ± 4.49 mm, while that of samples with hamartin
Breslow thickness and ulceration of melanoma lesions expression was 5.00 ± 3.74 mm (P = 0.364).
are important prognostic factors for melanoma. The overall Among the 91 patients with TNM stage data, the per-
ulceration rate in our cohort was 57.3% (47/82). The ulcera- centages of patients harbouring TSC1 mutations at stages
tion rates in mucosal melanoma arising from the head and I, II, III, and IV were 0% (0/16), 6.2% (1/16), 31.2%
neck, genitourinary tract, anorectum, and oesophagus were (5/16), and 62.5% (10/16), respectively, which were
53.8% (21/39), 61.9% (13/21), 58.8% (10/17), and 60.0% significantly different from those with wild-type TSC1
(3/5), respectively. Statistical differences were not found (P = 0.003) (Table 7). The frequency of stage III or IV in
for ulceration rates between mucosal melanoma with and patients harbouring mutations (83.3%) was significantly
without TSC1 mutations (P = 0.563) or between mucosal higher (P = 0.016) than stage III or stage IV in patients
melanoma with hamartin loss and expression (P = 0.260) with the wild-type gene (52.1%).
(Table 7).

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Journal of Cancer Research and Clinical Oncology (2018) 144:257–267 263

Table 7  Correlation of Clinical features TSC1 genotype Hamartin expression

TSC1 mutation and hamartin
a
expression to clinical features of Mutation WT P ­value Negative Positive P ­valuea
mucosal melanoma
Sex (female, %) 11/16,68.8 40/75,53.3 0.259 25/39,64.1 26/52,50.0 0.180
Age (median, year) 54.5 55 0.064 53 56 0.396
Ulceration (%) 9/14 (64.3) 38/68 (55.9) 0.563 17/34 (50.0) 30/48 (62.5) 0.260
Thickness (mm) 6.27 ± 3.55 5.19 ± 4.17 0.434 6.02 ± 4.49 5.00 ± 3.74 0.364
TNM stage (%)
I 0/16 1/75 (1.3) 0.003 0/39 1/52 (1.9) 0.624
II 1/16 (6.2) 36/75 (48.0) 11/39 (28.2) 8/52 (15.4)
III 5/16 (31.2) 14/75 (18.7) 14/39 (35.9) 23/52 (44.2)
IV 10/16 (62.5) 24/75 (32.0) 14/39 (35.9) 20/52 (38.5)
Anatomic site (%)
Head and neck 3 (18.8) 40 (53.3) 0.088 18 (46.2) 25 (48.1) 0.875
Genitourinary 6 (37.5) 16 (21.3) 9 (23.1) 13 (25.0)
Anorectum 5 (31.2) 15 (20.0) 10 (25.6) 10 (19.2)
Oesophagus 2 (12.5) 4 (5.3) 2 (5.1) 4 (7.7)
Other mutations
mTOR 1/16 (6.2) 11/75 (14.7) 0.620
BRAF 5/16 (31.2) 8/49 (16.3) 0.195
NRAS 6/48 (12.5) 2/16 (12.5) 1
CKIT 3/16 (18.8) 4/65 (6.2) 0.105
a
The unpaired t test was used for evaluation of age and thickness, Chi-square tests were used for evaluation
of sex, ulceration, subtypes and other gene mutations, Kruskal–Wallis test was used for evaluation of stages

Prognostic significance of TSC1 mutations for the OS no significant difference between patients with or without
of mucosal melanoma patients hamartin expression (P = 0.807) (Fig. 2c). All the clinico-
pathologic parameters, TSC1 mutation, mTOR mutation
We further analysed the relationship between all the clin- and hamartin expression were then included in multivari-
icopathologic parameters and OS. The survival data were ate Cox regression analysis, we found both TSC1 muta-
collected when patients were diagnosed with mucosal mela- tion (P = 0.005) and mTOR mutation (P = 0.024) status
noma and followed up until December 2016. The median may definitely serve as independent prognostic factor for
follow-up period was 31 months (range 3–168 months). OS (Table 8). In addition, we also analysed the correlation
Stage, ulceration, and thickness of melanoma lesions are between the activation of the two downstream molecules
important clinical features of melanoma and are known to S6RP or 4E-BP1 and patients’ OS. We found that no sig-
be prognostic factors for the prediction of the outcomes of nificant difference between the survival times of patients
melanoma (Balch et al. 2001; Manola et al. 2000). But in our with different S6RP or 4E-BP1 phosphorylation status (data
cohort, sex, age, thickness and ulceration were not of prog- not shown).
nostic significance for mucosal melanoma patients (Table 8).
However, the OS of patients at advanced stages (III and IV)
was significantly shorter than those at an early stage (I and Discussion
II, P = 0.003), which was consistent with previous studies
(Chi et al. 2011). Then, we further analysed the prognostic Being a relatively rare clinical entity in most areas of the
significance of TSC1 mutations and hamartin expression for world, mucosal melanoma has not been well investigated.
OS. We found that the median survival time for patients Moreover, the prognosis of mucosal melanoma is much
with TSC1 mutations (24 months, 95% CI: 18.97–29.03) worse than that of cutaneous melanomas (Chang et al.
was significantly shorter than that for patients with wild-type 1998; Chi et al. 2011). Genetic selection of specific targets
TSC1 (34 months, 95% CI 24.78–43.22, P = 0.007) (Fig. 2a). might be useful for the establishment of therapeutic strate-
In addition, patients with mTOR mutations also inclined to gies for mucosal melanoma patients. Recently, the PI3K/
have a worse survival (16 months, 95% CI 12.65–19.35) Akt/mTOR pathway has drawn considerable attention as an
than patients without mTOR mutation (33 months, 95% alternative therapeutic target (Madhunapantula et al. 2011).
CI 27.40–38.60, P = 0.004) (Fig. 2b). However, there was As a negative upstream regulator of mTOR, TSC1 might be

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264 Journal of Cancer Research and Clinical Oncology (2018) 144:257–267

Table 8  Univariate and Parameter Category OS

multivariate regression model
for overall survival Univariate Multivariate
a
HR 95% CI P ­value HR 95% CI P ­valueb

Gender Female 1
Male 1.437 0.92–2.40 0.116
Age (years) < 60 1
≥ 60 0.679 0.42–1.15 0.161
Ulceration Yes 1
No 1.254 0.76–2.16 0.372
Thickness (mm) <4 1
≥4 1.107 0.58–2.13 0.759
TNM stage I II 1
III IV 1.985 1.31–3.35 0.003
Anatomic site Head and neck 1
Others 0.850 0.53–0.34 0.476
TSC1 mutation WT 1 1
Mut 2.117 1.36–5.92 0.007 2.988 1.38–6.46 0.005
mTOR mutation WT 1 1
Mut 2.424 1.55–9.57 0.004 2.659 1.14–6.21 0.024
Hamartin Positive 1
Negative 1.059 0.66–1.70 0.807

CI confidence interval, Mut mutation, WT wild type

a
P values were derived from Kaplan–Meier analysis
b
P values were derived from Cox regression analysis

Fig. 2  Overall survival of melanoma patients in relation to TSC1 mutations (a), mTOR mutation (b), and hamartin expression (c)

a promising target for treating melanoma, especially intrac- in melanoma, and the mutation spectrum of TSC1 in mucosal
table mucosal melanoma. Disruption of the TSC1–TSC2 melanoma has not been reported before. TSC1 mutation rate
protein complex can result in the upregulation of signal in skin melanoma was only 4.2% according to TCGA pro-
transduction through the mTORC1 pathway, resulting in a gram. In our research, the rate of TSC1 mutations in mucosal
constitutive growth phenotype (Huang and Manning 2008). melanoma was 17.6%, which is significantly higher than that
According to TCGA, TSC1/TSC2 mutations have been iden- reported in skin melanoma thus far. Furthermore, by fur-
tified at a low frequency in a wide variety of cancers, with ther analyzing exome sequencing data in 25 different can-
the highest mutation rate being in bladder cancer, up to 16% cer types from TCGA, a total of 84 mutations were found.
(Lawrence et al. 2014; Platt et al. 2009). However, TSC1 The most common mutation types were missense (69.0%,
gene aberrations have not been comprehensively investigated 58/84) and truncating (29.8%, 25/84), including nonsense,

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Journal of Cancer Research and Clinical Oncology (2018) 144:257–267 265

nonstop, frameshift deletion, frameshift insertion and splice inactivates BRAF (Im et al. 2002; Karbowniczek et al.
site mutation, and the remaining was in-frame mutation 2004). Activation of mTOR leads to increased protein syn-
(1.2%, 1/84). However, in our research, nonsense mutation thesis through phosphorylation of p70S6K and 4E-BP1.
was at the highest rate (93.7%, 15/16) and the remaining one These two opposing effects may counterbalance each other,
was in-frame deletion (7.1%, 1/16), no truncating mutation naturally limiting Rheb’s ability to induce tumour growth
was founded. The mutation pattern of mucosal melanoma in cells expressing BRAF and contributing to the fact that
seemed to be quite different from that of other cancer types. the vast majority of tumours in TSC patients are benign.
Therefore, the comprehensive, our study is of great signifi- In mucosal melanoma, NRAS/BRAF mutations resulted in
cance for patients with mucosal melanomas. the activation of BRAF and downstream pathway, which
The significance of hamartin expression and TSC1 muta- counteracted the suppression of BRAF by TSC1 inactive
tions in melanoma prognosis has not been evaluated before. mutation. The balance between the two opposing effects
In this study, we found that patients with TSC1 mutations by Rheb was broken and then tumorigenesis was launched.
tend to have a worse outcome than patients with wild type This was consistent with what was found in our research
and TSC1 mutation may serve as a worse prognostic factor that TSC1 and BRAF/NRAS mutations are not mutually
in mucosal melanoma according to multivariate Cox analy- exclusive in mucosal melanoma.
sis, but due to our small cohort size, it should be further Previous studies in bladder cancer and hepatocellular
validated in more patients. Loss of hamartin expression has carcinomas demonstrated that TSC mutations were sensi-
been suggested as an adverse prognostic factor for survival tive to treatment with mTOR inhibitors (Guo et al. 2013;
in gastric carcinoma (Byeon et al. 2014). But in our research, Ho et al. 2016). An earlier study in metastatic urothelial
hamartin expression is unrelated to survival time in mucosal cancer showed that patients harbouring TSC1 mutation
melanoma. could benefit from everolimus treatment, an inhibitor of
The mTOR pathway was found to be activated in patients mTOR (Iyer et al. 2012). Analysis of urothelial carci-
with TSC1 mutations or hamartin loss in our study. The noma samples from patients who had clinical benefit from
expression rate of phospho-S6RP was significantly higher everolimus therapy showed that four of five patients with
in samples with TSC1 mutations or hamartin loss than those TSC1 mutation experienced objective tumour shrinkage,
with TSC1 wild-type or hamartin expression. However, the compared with one of nine without TSC1 mutation (Iyer
expression of hamartin was not correlated with TSC1 muta- et al. 2012).Given that the TSC1 mutations identified in
tion. Hence, the inactive TSC1 mutations identified in our our study could activate the mTOR pathway, it can be sur-
findings might mainly affect the mTOR/p70S6K pathway but mised that mucosal melanoma patients harbouring TSC1
not via the downregulation of TSC1 expression which might mutations might benefit from mTOR pathway inhibitors.
be attributable to mutation distribution according to TSC1 Phase II clinical trials of mTOR inhibitors in advanced
domains. The most frequently affected region of hamartin by melanomas have shown limited effects without selecting
the mutations (31.3%, 5 of 16) in our study was the coiled- patients with somatic mutation (Dronca et al. 2014; Rao
coil region (730–996), followed by the tuberin-binding et al. 2006). Screening of TSC1 mutation will aid in the
domain (302–430) (18.8%, 3/16). Coiled-coil regions are identification of patients most likely to respond to mTOR
often responsible for protein–protein interactions. The pro- pathway inhibitors. Further studies are required to func-
teins coded by TSC1 and TSC2 bind to each other via their tionally investigate whether the somatic TSC1 mutations
respective coiled-coil domains to form the complex (van could activate mTOR/p70S6K pathway in mucosal mela-
Slegtenhorst et al. 1998). Additionally, Nellist et al. iden- noma cells and whether these TSC1 mutants were sensitive
tified a tuberin-binding domain that is not located within to mTOR pathway inhibitors.
the coiled-coil region, but spans residues 302–430 (Nellist In summary, our research presents the first systematic
et al. 2001). Therefore, mutations in these two regions might analysis of TSC1 somatic mutations in mucosal melanoma.
destabilise the formation of hamartin and tuberin, leading to TSC1 mutations occurred in 17.6% of the mucosal mela-
deregulated expression patterns in the mTOR pathway and noma cases analysed and could activate the downstream
abnormal production of the following products. Consider- mTOR pathway. Our findings highlight the close involve-
ing that these mutations affected domains responsible for ment of TSC1 mutations in mucosal melanoma and draw
TSC2-binding, mutations of TSC1 in mucosal melanoma further attention to personalised therapeutic options for a
might not result in the downregulation of protein level, but novel molecular subset of patients with TSC1 mutations,
instead affect the structure of the hamartin–tuberin complex. which can be achieved by the inhibition of mTOR signalling.
In our research, we found that TSC1 and BRAF/NRAS
mutations are not mutually exclusive. As the target Acknowledgements The authors thank the staff in the Department of
Pathology of our hospitalfor help in collection and pathologic analysis
of tuberin’s (TSC2) GTPase activating protein (GAP) of tissue samples.
domain, Rheb has a dual role: it activates mTOR and

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266 Journal of Cancer Research and Clinical Oncology (2018) 144:257–267

Compliance with ethical standards Dronca RS et al (2014) Phase II study of temozolomide (TMZ)
and everolimus (RAD001) therapy for metastatic melanoma
a North Central Cancer Treatment Group Study, N0675. Am
Funding This study was funded by grants from the National Natural
J Clin Oncol-Canc 37:369–376. https://doi.org/10.1097/
Science Foundation of China (51402264, 81672696), Beijing Talents
COC.0b013e31827b45d4
Fund (2016000021223ZK18), Beijing Municipal Natural Science
Furney SJ et al (2013) Genome sequencing of mucosal melanomas
Foundation (7152033), Baiqianwan Talents Project, Beijing Municipal
reveals that they are driven by distinct mechanisms from cutane-
Administration of Hospitals Clinical medicine Development of special
ous melanoma. J Pathol 230:261–269. https://doi.org/10.1002/
funding support (ZYLX201603), and Beijing Municipal Science &
path.4204
Technology Commission (Z151100003915074).
Guo Y, Chekaluk Y, Zhang J, Du J, Gray NS, Wu CL, Kwiatkowski
DJ (2013) TSC1 involvement in bladder cancer: diverse effects
Conflict of interest The authors declare that they have no conflict of and therapeutic implications. J Pathol 230:17–27. https://doi.
interest. org/10.1002/path.4176
Hayward NK et al (2017) Whole-genome landscapes of major. mela-
Ethical approval All procedures performed in studies involving human noma subtypes Nature 545:175–180. https://doi.org/10.1038/
participants were in accordance with the ethical standards of the Medi- nature22071
cal Ethics Committee of the Beijing Cancer Hospital & Institute and Ho DW et al. (2016) TSC1/2 mutations define a molecular subset of
with the 1964 Helsinki declaration and its later amendments or com- HCC with aggressive behaviour and treatment implication. Gut.
parable ethical standards. For this type of study formal consent is not https://doi.org/10.1136/gutjnl-2016-312734
required. Huang J, Manning BD (2008) The TSC1-TSC2 complex: a molecular
switchboard controlling cell growth. Biochem J 412:179–190.
Informed consent Informed consent was obtained from all individual https://doi.org/10.1042/BJ20080281
participants included in the study. Im E et al (2002) Rheb is in a high activation state and inhibits B-Raf
kinase in mammalian cells. Oncogene 21:6356–6365. https://
doi.org/10.1038/sj.onc.1205792
Iyer G et al (2012) Genome sequencing identifies a basis for everoli-
mus sensitivity. Science 338:221. https://doi.org/10.1126/
science.1226344
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