J Neuropathol Exp Neurol
Copyright ˘ 2013 by the American Association of Neuropathologists, Inc.
Vol. 72, No. 9
September 2013
pp. 814Y821
ORIGINAL ARTICLE
Molecular and Clinical Risk Factors for Recurrence of Skull
Base Chordomas: Gain on Chromosome 2p, Expression
of Brachyury, and Lack of Irradiation Negatively
Correlate With Patient Prognosis
Yohei Kitamura, MD, Hikaru Sasaki, MD, PhD, Tokuhiro Kimura, MD, PhD, Tomoru Miwa, MD, PhD,
Satoshi Takahashi, MD, PhD, Takeshi Kawase, MD, PhD, and Kazunari Yoshida, MD, PhD
INTRODUCTION
Abstract
Chordomas are invasive tumors that develop from notochordal
remnants and frequently occur in the skull base. The T gene and its
product (brachyury) have recently been suggested to play an important role in chordoma progression. To date, few studies have
investigated the relationship between the molecular/genetic characteristics of chordoma and patient prognosis. We analyzed 37 skull base
chordomas for chromosomal copy number aberrations using comparative genomic hybridization, brachyury expression by immunohistochemistry, and T gene copy number by fluorescence in situ hybridization.
The results of these molecular analyses and clinical parameters were
compared with the patients’ clinical courses. Univariate analyses using the
log-rank test demonstrated that losses on chromosome 1p and gains on 1q
and 2p were negatively correlated with progression-free survival, as were
factors such as female sex, partial tumor removal, lack of postoperative
irradiation, and high MIB-1 index. Expression of brachyury and copy
number gain of the T gene were also significantly associated with shorter
progression-free survival. Multivariate analysis using the Cox hazards
model showed that lack of irradiation, gain on chromosome 2p, and
expression of brachyury were independently associated with a poor
prognosis. Our results suggest that brachyury-negative chordomas
are biologically distinct from brachyury-positive chordomas and that
T/brachyury might be an appropriate molecular therapeutic target
for chordoma.
Key Words: Brachyury, Chromosome 2p, Comparative genomic
hybridization, Fluorescence in situ hybridization, Skull base chordoma,
T gene.
From the Departments of Neurosurgery (YK, HS, TM, ST, TKawase , KY),
and Pathology (TKimura), Keio University School of Medicine, Tokyo,
Japan.
Send correspondence and reprint requests to: Hikaru Sasaki, MD, PhD,
Department of Neurosurgery, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; E-mail: hsasaki@
a5.keio.jp
The authors have no personal, financial, or institutional interest in any of the
drugs, materials, or devices described in this article.
Supported by the Japan Society for the Promotion of Science KAKENHI
Grant Number 24791519 and Keio University Grant-in-Aid for Encouragement of Young Medical Scientists.
Supplemental digital content is available for this article. Direct URL citations
appear in the printed text and are provided in the HTML and PDF versions
of this article on the journal’s Web site (www.jneuropath.com).
814
Chordomas are invasive tumors that develop from notochordal remnants and frequently occur in the sacrum, vertebral
body, and skull base. Skull base chordoma, which comprises
32% of all chordomas (1) and less than 0.1% to 0.5% of primary intracranial CNS tumors (2, 3), tends to show low-grade
malignancy. It has a high frequency of recurrence, however,
partly because complete resection is difficult. At present, the
mainstay of treatment is maximum resection with skull base
surgery, followed by irradiation of the residual tumor using
conventional, carbon ion, and proton beam radiotherapies
(4Y6). Antineoplastic and molecule-targeted agents have been
tried in advanced cases, but their effectiveness has been uncertain (7, 8).
Several molecular and clinical studies have been conducted on skull base chordomas (4, 5, 9, 10). According to
these studies, poor prognosis is associated with biologic
factors (e.g. loss on chromosomes 1p and 9p and a high proliferative index), therapy-related factors (e.g. lack of postoperative irradiation and partial tumor removal), and patient
characteristics (e.g. advanced age). However, most of the
previous genetic studies analyzed only several specific loci
using loss of heterozygosity (LOH) and fluorescence in situ
hybridization (FISH) assays. To our knowledge, there have
been no whole-genome studies (other than karyotyping) with
statistical analysis in relation to prognosis. Moreover, there
have been no studies investigating the relationship of both
molecular and clinical parameters with patient prognosis.
The T gene, encoded on 6q27, has recently been suggested to play an important role in tumorigenesis and progression of chordoma. Brachyury, the protein product of T,
is expressed in the majority of chordomas but rarely in other
tumors (11Y13). A germline alteration at the T gene site is
associated with familial chordoma (14), and T amplification
is also found in some sporadic chordomas (15). Suppression
of brachyury expression halts cell proliferation in chordoma
cell lines, whereas overexpression of brachyury enhances
cell proliferation in the cell lines (15Y18). However, there are
no previous reports investigating the relationship between
brachyury expression/T gene copy number gain (CNG) and
the actual clinical behavior of the tumor.
J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
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J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
In the present study, we performed whole-genome
analysis using comparative genomic hybridization (CGH) of
37 skull base chordomas. We also investigated T gene status
by immunohistochemistry (IHC) and FISH, thereby providing
the first comprehensive analysis of both molecular and clinical
parameters in relation to the prognosis of patients with skull
base chordomas.
MATERIALS AND METHODS
Tumor Samples
We used formalin-fixed paraffin-embedded tissues of
the tumor specimens resected from 37 patients with skull
base chordomas at Keio University Hospital between 1993
and 2011. This work was approved by the institutional review
board of Keio University Hospital (Reference No. 20120282).
The diagnosis of chordoma was based on hematoxylin
and eosin staining. For some specimens that were difficult
to distinguish from chondrosarcoma, IHC for epithelial markers
such as cytokeratin AE1/AE3 and epithelial membrane antigen
was also performed for histologic diagnosis. Clinical follow-up
data were available for all patients.
IHC for MIB-1
Some previous studies have implied that the MIB-1
index might be associated with chordoma prognosis (9).
Immunohistochemistry for MIB-1 was performed on formalinfixed paraffin-embedded sections, with antigen retrieval by
microwave irradiation. The sections were incubated with mouse
monoclonal antiYKi-67 antibody (MIB-1, 1:200; Dako, Glostrup,
Denmark). For quantitation of immunopositive nuclei, the
staining indices were analyzed in a total of more than 1,000
tumor cells from more than 3 areas, showing the typical
appearance of each tumor (19). For statistical analysis, the
boundary of high or low MIB-1 index was placed at 5%,
referring to the previous report (9).
Comparative Genomic Hybridization
Comparative genomic hybridization analysis was performed according to a previously described protocol (20). In
brief, tumor DNA was extracted from microdissected pieces
of formalin-fixed paraffin-embedded tissue and amplified by
degenerate oligonucleotide primed-polymerase chain reaction.
During tissue microdissection, the consecutive sections (stained
with hematoxylin and eosin) were inspected by a pathologist
(T.K.), and nontumoral tissue was excluded. Tumor DNA was
labeled with another degenerate oligonucleotide primedpolymerase chain reaction using digoxigenin-11-dUTP
(Roche, Mannheim, Germany), and the reference DNA was
amplified from 50 ng of normal male or female DNA and
labeled with biotin-dUTP (Roche). The probe mixture was denatured and hybridized to normal metaphase spreads (Vysis,
Downers Grove, IL). The unhybridized probes were washed
out, and the metaphase spread was incubated with fluorescein
isothiocyanateYconjugated anti-digoxigenin antibody (Roche)
and rhodamine-conjugated avidin (Roche). The preparations
were washed and counterstained with 4¶,6-diamidino-2phenylindole in antifade solution. Red, green, and blue images
Risk Factors in Skull Base Chordoma
were acquired, and the ratios of fluorescence intensity along
the chromosomes were quantitated using the CytoVision
analysis system (Applied Imaging, San Jose, CA).
IHC for Brachyury
After antigen retrieval in citrate buffer (pH 6) for
10 minutes by microwave irradiation, paraffin sections were
incubated with rabbit anti-brachyury antibody (H210, 1:400;
Santa Cruz Biotechnology, Santa Cruz, CA). Brachyury expression was considered positive when the nucleus was stained
strongly and diffusely and negative otherwise, as previously
described (11). Evaluation was conducted by 2 of the authors
(Yohei Kitamura, Tokuhiro Kimura) one of whom was the
pathologist (Tokuhiro Kimura).
Fluorescence In Situ Hybridization
The probes generated from the corresponding clones from
a library of human genomic clones (clone no. GSP3023A02)
were provided by GSP Laboratory Inc. (Kawasaki, Japan) (Figure,
Supplemental Digital Content 1, http://links.lww.com/NEN/A479).
The probe for the T gene was labeled with Texas Red, and the
probe for the specific sequence near the centromere of chromosome 6p (6p12.1/660kb, CEN6p) was labeled with fluorescein
isothiocyanate. The probes were successfully tested on the slides
with peripheral blood mononuclear cells in metaphase.
Sections were cut at 4-Km thickness. After boiling in
pretreatment solution (GSP Laboratory Inc.) for 30 minutes,
the slides were rinsed in distilled water and washed in 2
salineYsodium citrate buffer (SSC) twice for 5 minutes. The
slides were digested in protease solution (GSP Laboratory
Inc.) at 37-C for 15 minutes, rinsed twice with 2 SSC, and
dehydrated with a 70%/85%/100% ethanol series. The probes
(10 KL) were applied, and the slides were denatured on a hot
plate at 75-C. The sections were covered with cover glass
and rubber cement and incubated at 37-C for approximately
72 hours in a humid chamber. After hybridization, the slides
were washed with 2 SSC/0.3% NP-40 and then 2 SSC at
room temperature for 5 minutes. The nuclei were counterstained with 4¶,6-diamidino-2-phenylindole, and signals were
visualized by fluorescence microscopy. The signals were
counted in 50 nuclei of the tumor cells. Referring to the previous literature (15) and the HER2 criteria for breast cancer,
the most established FISH criteria in the clinical field (21),
we judged the result to be positive when the T/CEN6p ratio
was greater than 2.0 or the average T signal per nucleus was
greater than 4.0 for T gene CNG.
Statistical Analyses
Progression-free survival (PFS) was defined as a period
from the date of initial surgery until the first day of treatment
for recurrence. Progression-free survival for patients with no
recurrence was a period from the date of initial surgery until
the last follow-up date. Statistical analysis was performed using
JMP version 8.0.1 (SAS Institute, Cary, NC). The KaplanMeier method and log-rank test were used to evaluate the
relationship between PFS and molecular results, including
frequently observed chromosomal copy number alterations
(CNAs) (detected in Q5 tumors), brachyury expression, T gene
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815
J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
Kitamura et al
(18.9%) showed a high proliferative index of more than 5%
(Table 1).
TABLE 1. Clinical Parameters of 37 Chordoma Cases
Factors
n (%)
22 (59)
15 (41)
Statistical Analyses With Clinical Factors and
MIB-1 Index
Male
Female
Yes
No
17 (46)
20 (54)
19 (51)
18 (49)
The results of the univariate analysis using log-rank test
revealed that high MIB-1 index (p G 0.001), partial tumor removal (p = 0.029), lack of postoperative radiotherapy (p = 0.014),
and female sex (p = 0.036) were significantly correlated with
shorter PFS (Table 2). Age was not significantly associated
with PFS.
Gross total removal
Subtotal or partial removal
Yes
No
e5
95
8 (22)
29 (78)
17 (46)
20 (54)
30 (81)
7 (19)
940
e40
Age, years
Mean age, 43.6 years
(range, 10Y75 years)
Sex
Recurrence
Median time to recurrence,
20 months (range, 4Y68 months)
Extent of resection
Postoperative irradiation
MIB-1 index, %
Comparative Genomic Hybridization
Mean MIB-1 index, 4.2%
(range, 0.2%Y19.0%)
CNG, and MIB-1 index. Clinical data, including sex, age,
extent of tumor removal, and use of postoperative irradiation,
were also evaluated in relation to PFS. For the factors with a
value of p G 0.05 in univariate analysis, multivariate analysis
using the Cox proportional hazards model was applied to
examine independent prognostic factors. Because there were
too few cases with events, it was difficult to perform statistical
analysis with overall survival.
Copy number alterations were observed in 24 cases
(64.9%). Frequent CNAs (observed in 95 patients) were
losses on chromosomes 1p, 3p, 9, 10q, 13q, 14q, and 18q and
gains on 1q, 2p, 6q, 7, 17q, and 19q (Fig. 1). The most frequent CNA was gain on chromosome 7, which was observed
in 10 cases (27.0%); the next most frequent was a gain on 1q,
which was observed in 9 cases (24.3%). The log-rank test
evaluating the relationship between PFS and frequent CNAs
revealed that loss on 1p (p = 0.034) and gain on 1q and 2p
(p = 0.001 and p G 0.001, respectively) were significantly
associated with PFS (Table 2).
IHC for Brachyury
Immunohistochemistry for brachyury was positive in
30 cases (81.1%) and negative in 7 cases (18.9%) (Fig. 2). All
cases with gain on 6q were also positive for brachyury on
TABLE 2. Results of Univariate Analyses Using Log-Rank Test
Factor
RESULTS
Patient Characteristics
The 37 patients with skull base chordomas consisted
of 17 males and 20 females, with a mean age of 43.6 years
(range, 10Y75 years) (Table 1; Table, Supplemental Digital
Content 2, http://links.lww.com/NEN/A480). The mean follow-up
period was 62.4 months (range, 0Y146 months). Recurrence
was observed in 19 cases (51.4%), and the median time from
surgery until recurrence was 20 months (range, 4Y68 months).
Three patients died during our follow-up. The extent of tumor
removal was defined on the basis of operation records and
postoperative magnetic resonance images. Gross total removal
(defined by no evidence of residual tumor on postoperative
magnetic resonance images or by the description of macroscopically complete resection in the operation record) was
achieved in 8 cases (21.6%); partial removal (defined as resection of G90% of the tumor) was achieved in 29 cases (78.4%).
There were no cases of subtotal removal (defined as resection of 990% of the tumor). Postoperative irradiation was
performed in 17 cases (45.9%). Irradiation included conventional, carbon ion, and proton beam radiotherapies. Previous
studies indicate that there are no significant differences between
these radiotherapies when applied to skull base chordomas (5).
The mean MIB-1 index was 4.2% (0.2%Y19.0%), and 7 cases
816
n (%)
Sex (female)
Age (940 years)
Extent of removal (STR/PR)
Radiation (absent)
MIB-1 index (95%)
j1p32j34.3
+1q42jter
+2p
V3p14.2j22
+6q
+7pterjq31
j9p21j23
j9q22
j9q31j33
j10q21j22
j13qcenj14
j14q13j24
+17q23jter
j18q12.3j21
+19q13.2
IHC of brachyury (positive)
FISH of T gene (positive)
21 (57)
22 (59)
29 (78)
16 (43)
7 (19)
8 (22)
6 (16)
5 (14)
5 (14)
5 (14)
10 (27)
5 (14)
6 (16)
6 (16)
7 (19)
7 (19)
8 (22)
7 (19)
7 (19)
5 (14)
30 (81)
6 / 22 (27)
p
0.036*
0.813
0.029*
0.014*
G0.001*
0.034*
0.001*
G0.001*
0.333
0.343
0.241
0.150
0.929
0.271
0.374
0.173
0.147
0.255
0.434
0.245
0.034*
0.023*
* p G 0.05.
FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; PR, partial
removal; STR, subtotal removal.
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Copyright © 2013 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
Risk Factors in Skull Base Chordoma
FIGURE 1. Summary of chromosomal aberrations in 37 chordomas detected by comparative genomic hybridization. Lines to the
left of each idiogram represent regions of relative loss; lines to the right represent regions of relative gain.
IHC. Interestingly, CNA was infrequent in the 7 cases with
brachyury-negative chordomas, and tumors in 3 of these cases
showed no CNAs (Table, Supplemental Digital Content 2,
http://links.lww.com/NEN/A480).
The log-rank test for IHC of brachyury and PFS showed
significant intercorrelation (p = 0.034; Table 2). Brachyury
expression was significantly associated with shorter PFS
(Fig. 3A).
FIGURE 2. Immunohistochemistry for brachyury. (A) A case positive for brachyury. (B) A case negative for brachyury. Inset shows
the consecutive section stained with hematoxylin and eosin.
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J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
Kitamura et al
FIGURE 3. (A) Kaplan-Meier survival plot showing the progression-free survival (PFS) rates in cases with (+) or without (-) brachyury
expression, as detected using immunohistochemistry. (B) Kaplan-Meier survival plot showing the PFS rates in cases with (+) or
without (-) copy number gain of the T gene.
FISH of the T Gene
T and CEN6p signals were reliably assessed in only 22
of the 37 specimens because of the strong background interference caused by the extracellular matrix (Table 3). T gene
CNG was observed in 6 cases (Fig. 4BYD; Table, Supplemental Digital Content 2, http://links.lww.com/NEN/A480).
Among the 4 cases with a gain on 6q revealed by CGH, 2 cases
were positive and the other 2 cases showed a minor CNG in the
FISH assay. On IHC, all 6 FISH-positive chordomas expressed
brachyury.
The log-rank test for PFS and FISH results showed significant intercorrelation (p = 0.023; Table 2). Progression-free
survival of the patients with skull base chordomas with T gene
CNG was significantly shorter than that of those without this
CNG (Fig. 3B).
ratio, 18.98 and 5.775, p = 0.030 and 0.043, respectively;
Table 4).
DISCUSSION
In the present study, we analyzed 37 skull base chordomas
for CNAs, proliferative activity, and the status and protein
product of the T gene and investigated the relationship of the
molecular and clinical factors with patient prognosis. We
Multivariate Analyses
Multivariate analyses were conducted with factors that
were significantly associated with recurrence in univariate
analyses, including sex, extent of tumor removal, use of irradiation, MIB-1 index, loss on chromosome 1p, gains on 1q
and 2p, and brachyury expression or T gene CNG. In the
analysis with brachyury expression, brachyury expression and
lack of irradiation showed trends toward association with
recurrence (hazard ratio, 5.480 and 2.675, p = 0.055 and
0.063, respectively; Table 4). On the other hand, if T gene
CNG instead of brachyury expression is included in the
multivariate analysis, gain on 2p and lack of irradiation are
significantly associated with the risk of recurrence (hazard
TABLE 3. Results of FISH of the T Gene
Average T Signals Per Nucleus
94.0
e4.0
94.0
e4.0
818
T/CEN6p Ratio
No. Cases
92.0
92.0
e2.0
e2.0
3
0
3
16
FIGURE 4. Representative examples of fluorescent in situ hybridization (FISH) of the T gene. (A) FISH with T gene (red)/
CEN6p (green) probes on the metaphase spread of a normal
lymphocyte showing red signals at 6q27 and green signals at
6p regions near the centromere. Examples of chordomas with
T gene copy number gains of disomy (B), polysomy (C), and
amplification (D).
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J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
Risk Factors in Skull Base Chordoma
TABLE 4. Results of Multivariate Analysis Using the Cox Proportional Hazards Model
Univariate analysis
Factor
Gender (female)
Extent of recestion (STR/PR)
Irradiation (absent)
MIB-1 index (95%)
j1p
+1q
+2p
IHC of brachyury (positive)
FISH of T gene (positive)
Multevariate analysis (brachyury IHC)
Multivariate analysis (T gene FISH)
p
HR
95% CI
p
HR
95% CI
p
0.036
0.029
0.014
G0.001
0.034
0.001
G0.001
0.034
0.023
1.796
2.707
2.675
1.890
0.577
1.621
4.551
5.480
0.643 to 5.615
0.561 to 20.23
0.951 to 8.416
0.267 to 10.93
0.088 to 2.706
0.157 to 15.04
0.381 to 50.67
0.970 to 103.7
0.269
0.226
0.063
0.496
0.503
0.675
0.227
0.055
1.372
0.283
5.775
2.113
0.395
1.057
18.98
0.249 to 10.51
0.017 to 6.932
1.057 to 56.45
0.365 to 15.50
0.018 to 8.642
0.044 to 15.11
1.346 to 430.6
0.723
0.378
0.043
0.400
0.530
0.968
0.030
2.647
0.209 to 32.97
0.430
STR: subtotal removal, PR: partial removal, IHC: immunohistochemistry, FISH: fluorescence in situ hybridization.
found that the factors that significantly increased the risk
of recurrence included high MIB-1 index, loss on 1p, gain
on 1q and 2p, brachyury protein expression, T gene CNG,
female sex, partial tumor removal, and lack of irradiation.
Among those factors, gain on 2p, lack of irradiation, and
expression of brachyury seemed to independently predict
the risk of recurrence. This is the first report suggesting that
T gene abnormality is a negative prognostic factor for skull
base chordomas.
There are many studies in the literature that have performed
genetic analysis of chordoma. Frequently found genetic abnormalities are the losses on chromosomes 1p, 3, 4, 9, 10, 13, 14, and
18, as well as gains on 1q and 7 (22Y27), and those aberrations
were also frequently observed in the present study. Of these
aberrations, the most intensely investigated are those on the
chromosome arm 1p. Several investigators have suggested
(mostly on the basis of LOH or FISH results) that 1p36 might
be a putative tumor suppressor locus of skull base chordoma
and found that the loss on 1p36 is associated with poor
prognosis in those tumors (28Y31). Sawyer et al (31) reported isochromosome 1q and monosomy 13 as frequent structural abnormalities in skull base chordomas using spectral
karyotyping, supporting the idea of the tumor suppressor
locus on 1p. Horbinski et al (9) suggested that a Ki67 proliferative index of 5% or higher and 9p LOH are significantly
associated with shorter survival in patients with skull base
chordomas; using standard karyotyping, Almefty et al (27)
reported that aberrations of chromosomes 3, 4, 12, 13, and 14
correlate with frequent recurrence and decrease the duration
of survival. However, most of these genetic studies were
either analyses of only a few specific loci using the LOH/FISH
assay (9, 28, 30, 32, 33) or whole-genome studies without
statistical analysis in relation to prognosis (22, 23, 31). Moreover, all the previous genetic analyses relating to patient
prognosis did not include clinical parameters such as irradiation
and extent of tumor removal. To our knowledge, this is the first
report that related both the results of whole-genome analysis
and clinical parameters to the prognosis of patients with skull
base chordomas. Our analysis reveals that loss on 1p and gain
on 1q and 2p are associated with an increased risk of recurrence. Because gain on 1q may be shown as a relative loss
on 1p by the LOH assay, our results might have underscored
the importance of loss on 1p in association with aggressive
chordomas. Importantly, the present study is the first to suggest
that gain on 2p might be associated with poor prognosis in
skull base chordomas. Interestingly, gain on 2p has been
suggested to be associated with progression of chronic lymphocytic leukemia in some recent reports (34, 35). Thus,
oncogenes encoded on 2p such as MYCN, REL, and ALK might
have a role in aggressive chordomas.
Brachyury was first discovered in mice as a protein that
plays an important role in notochord development and the
formation of posterior mesodermal elements during the fetal
period (36), and it was later clarified that its homolog is also
present on human chromosome 6q27 (37). Brachyury has
been reported as a highly specific marker for chordoma and
is usually not expressed in other tumors, including chondrosarcoma (11Y13). T duplication was detected in the
germline of patients with familial chordoma (14), and T amplification is also found in some sporadic chordomas (15).
Moreover, several in vitro studies conducted using chordoma
cell lines have suggested that brachyury plays an important
role in chordoma progression; therefore, T is considered an
‘‘oncogene’’ (15Y18). From the clinical point of view, this
is the first report that investigated T gene status in relation to
patient prognosis. Indeed, in the present study, brachyury
expression and T gene CNG were associated with an increased
risk of recurrence, and PFS of the patients with chordomas
expressing brachyury and/or T gene CNG was significantly
shorter than that of the individuals without those aberrations.
Chordomas without brachyury expression on IHC comprise
2% to 17% of all chordomas (11Y13, 38); in our cohort, they
constituted 18.9%. Our study suggests that those brachyurynegative chordomas are biologically distinct from brachyurypositive tumors. TBX2, a T gene family member, acts as an
oncogene repressing the tumor suppressor CDKN2A in various
cancers including melanoma, breast cancer, and leukemia;
Nelson et al (18) suggested the possibility of epidermal growth
factor receptor activation by brachyury. These pathways might
be implicated in the regulation of the oncogenic activity of
brachyury. Curiously, CNA was infrequent in brachyurynegative chordomas analyzed by CGH. Moreover, the average
of MIB-1 index in brachyury-negative chordomas was lower
than brachyury-positive chordomas (1.47% vs. 4.78%). We
speculate that brachyury might be associated with chromosomal
instability and proliferative activity.
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J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
Kitamura et al
Lack of postoperative irradiation and high MIB-1 index
have previously been reported to be correlated with poor
prognosis of patients with skull base chordomas (5, 9). A
few reports also suggested a trend toward correlation of
incomplete tumor removal with poor prognosis (10, 39).
These findings were confirmed in our study by univariate or
multivariate analyses. Forsyth et al (10) reported that age
(G40 years) is strongly associated with longer overall survival
and disease-free survival, but the present study demonstrated
no correlation between age and recurrence. In contrast to some
previous reports that indicated lack of statistical relationship
between sex and prognosis (4, 40), however, our study suggested
that female sex might constitute a risk for shorter PFS.
In conclusion, our comprehensive analysis of both
molecular and clinical factors suggests that high MIB-1 index,
loss on 1p, gain on 1q and 2p, brachyury expression, T gene
CNG, female sex, partial tumor removal, and lack of irradiation are associated with the risk of recurrence or shortening
of PFS in skull base chordomas. Chordomas with these
risk factors should be followed up carefully. Our results
also suggest that brachyury-negative chordomas are biologically distinct from brachyury-positive chordomas and that
T/brachyury might be an appropriate molecular target for
therapy of these neoplasms.
ACKNOWLEDGMENTS
We thank Ms. Naoko Tsuzaki and Ms. Kiyomi Koide
for their technical assistance. We also thank Dr. Masahiko
Maekawa (GSP Laboratory, Inc.) and Dr. Yoshifumi Okada
(Dokkyo Medical University) for their technical advices.
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Tumor Registry of Japan (1984Y2000). Neurol Med Chir (Tokyo) 2009;
49(Suppl):1Y96
3. CBTRUS. Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2004Y2007, 2011
4. Favre J, Deruaz JP, Uske A, et al. Skull base chordomas: Presentation of
six cases and review of the literature. J Clin Neurosci 1994;1:7Y18
5. Takahashi S, Kawase T, Yoshida K, et al. Skull base chordomas: Efficacy
of surgery followed by carbon ion radiotherapy. Acta Neurochir (Wien)
2009;151:759Y69
6. Bugoci DM, Girvigian MR, Chen JC, et al. Photon-based fractionated stereotactic radiotherapy for postoperative treatment of skull base chordomas.
Am J Clin Oncol 2012 [Epub ahead of print]
7. Geoerger B, Morland B, Ndiaye A, et al. Target-driven exploratory study
of imatinib mesylate in children with solid malignancies by the Innovative Therapies for Children with Cancer (ITCC) European Consortium.
Eur J Cancer 2009;45:2342Y51
8. Stacchiotti S, Marrari A, Tamborini E, et al. Response to imatinib plus
sirolimus in advanced chordoma. Ann Oncol 2009;20:1886Y94
9. Horbinski C, Oakley GJ, Cieply K, et al. The prognostic value of Ki-67,
p53, epidermal growth factor receptor, 1p36, 9p21, 10q23, and 17p13 in
skull base chordomas. Arch Pathol Lab Med 2010;134:1170Y76
10. Forsyth PA, Cascino TL, Shaw EG, et al. Intracranial chordomas: A
clinicopathological and prognostic study of 51 cases. J Neurosurg 1993;
78:741Y47
11. Jambhekar NA, Rekhi B, Thorat K, et al. Revisiting chordoma with
brachyury, a ‘‘new age’’ marker: Analysis of a validation study on 51
cases. Arch Pathol Lab Med 2010;134:1181Y87
820
12. Oakley GJ, Fuhrer K, Seethala RR. Brachyury, SOX-9, and podoplanin,
new markers in the skull base chordoma vs chondrosarcoma differential:
A tissue microarrayYbased comparative analysis. Modern Pathol 2008;
21:1461Y69
13. Vujovic S, Henderson S, Presneau N, et al. Brachyury, a crucial regulator
of notochordal development, is a novel biomarker for chordomas. J Pathol
2006;209:157Y65
14. Yang XR, Ng D, Alcorta DA, et al. T (brachyury) gene duplication confers
major susceptibility to familial chordoma. Nat Genet 2009;41:1176Y78
15. Presneau N, Shalaby A, Ye H, et al. Role of the transcription factor T
(brachyury) in the pathogenesis of sporadic chordoma: A genetic and
functional-based study. J Pathol 2011;223:327Y35
16. Hsu W, Mohyeldin A, Shah SR, et al. Generation of chordoma cell
line JHC7 and the identification of brachyury as a novel molecular target.
J Neurosurg 2011;115:760Y69
17. Fernando RI, Litzinger M, Trono P, et al. The T-box transcription factor
brachyury promotes epithelial-mesenchymal transition in human tumor
cells. J Clin Invest 2010;120:533Y44
18. Nelson AC, Pillay N, Henderson S, et al. An integrated functional genomics approach identifies the regulatory network directed by brachyury
(T) in chordoma. J Pathol 2012;228:274Y85
19. Sasaki H, Yoshida K, Ikeda E, et al. Expression of the neural cell adhesion molecule in astrocytic tumors: An inverse correlation with malignancy. Cancer 1998;82:1921Y31
20. Hirose Y, Aldape K, Takahashi M, et al. Tissue microdissection and
degenerate oligonucleotide primed-polymerase chain reaction (DOPPCR) is an effective method to analyze genetic aberrations in invasive
tumors. J Mol Diagn 2001;3:62Y67
21. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of
Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in
breast cancer. J Clin Oncol 2007;25:118Y45
22. Brandal P, Bjerkehagen B, Danielsen H, et al. Chromosome 7 abnormalities
are common in chordomas. Cancer Genet Cytogenet 2005;160:15Y21
23. Scheil S, Bruderlein S, Liehr T, et al. Genome-wide analysis of sixteen
chordomas by comparative genomic hybridization and cytogenetics of
the first human chordoma cell line, U-CH1. Genes Chromosomes Cancer
2001;32:203Y11
24. Le LP, Nielsen GP, Rosenberg AE, et al. Recurrent chromosomal copy
number alterations in sporadic chordomas. PLoS One 2011;6:e18846
25. Tallini G, Dorfman H, Brys P, et al.. Correlation between clinicopathological features and karyotype in 100 cartilaginous and chordoid tumours.
A report from the Chromosomes and Morphology (CHAMP) Collaborative Study Group. J Pathol 2002;196:194Y203
26. Hallor KH, Staaf J, Jonsson G, et al. Frequent deletion of the CDKN2A
locus in chordoma: Analysis of chromosomal imbalances using array
comparative genomic hybridisation. Br J Cancer 2008;98:434Y42
27. Almefty KK, Pravdenkova S, Sawyer J, et al. Impact of cytogenetic abnormalities on the management of skull base chordomas. J Neurosurg
2009;110:715Y24
28. Riva P, Crosti F, Orzan F, et al. Mapping of candidate region for
chordoma development to 1p36.13 by LOH analysis. Int J Cancer 2003;
107:493Y97
29. Miozzo M, Dalpra L, Riva P, et al. A tumor suppressor locus in familial
and sporadic chordoma maps to 1p36. Int J Cancer 2000;87:68Y72
30. Longoni M, Orzan F, Stroppi M, et al. Evaluation of 1p36 markers
and clinical outcome in a skull base chordoma study. Neuro Oncol 2008;
10:52Y60
31. Sawyer JR, Husain M, Al-Mefty O. Identification of isochromosome 1q
as a recurring chromosome aberration in skull base chordomas: A new
marker for aggressive tumors? Neurosurg Focus 2001;10:E6
32. Shalaby AA, Presneau N, Idowu BD, et al. Analysis of the fibroblastic
growth factor receptor-RAS/RAF/MEK/ERK-ETS2/brachyury signalling
pathway in chordomas. Mod Pathol 2009;22:996Y1005
33. Presneau N, Shalaby A, Idowu B, et al. Potential therapeutic targets for
chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway. Br J Cancer 2009;
100:1406Y14
34. Chapiro E, Leporrier N, Radford-Weiss I, et al. Gain of the short arm of
chromosome 2 (2p) is a frequent recurring chromosome aberration in
untreated chronic lymphocytic leukemia (CLL) at advanced stages. Leuk
Res 2010;34:63Y68
˘ 2013 American Association of Neuropathologists, Inc.
Copyright © 2013 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 72, Number 9, September 2013
35. Jarosova M, Urbankova H, Plachy R, et al. Gain of chromosome 2p in
chronic lymphocytic leukemia: Significant heterogeneity and a new recurrent dicentric rearrangement. Leuk Lymphoma 2010;51:304Y13
36. Beddington RS, Rashbass P, Wilson V. BrachyuryVa gene affecting mouse gastrulation and early organogenesis. Development 1992;
116(Suppl):157Y65
37. Edwards YH, Putt W, Lekoape KM, et al. The human homolog T of the
mouse T(Brachyury) gene: Gene structure, cDNA sequence, and assignment to chromosome 6q27. Genome Res 1996;6:226Y33
Risk Factors in Skull Base Chordoma
38. Tirabosco R, Mangham DC, Rosenberg AE, et al. Brachyury expression
in extra-axial skeletal and soft tissue chordomas: A marker that distinguishes chordoma from mixed tumor/myoepithelioma/parachordoma in
soft tissue. Am J Surg Pathol 2008;32:572Y80
39. al-Mefty O, Borba LA. Skull base chordomas: A management challenge.
J Neurosurg 1997;86:182Y89
40. Gao Z, Zhang Q, Kong F, et al. Fascin expression in skull base
chordoma: Correlation with tumor recurrence and dura erosion. Med
Oncol 2012;29:2438Y44
˘ 2013 American Association of Neuropathologists, Inc.
Copyright © 2013 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
821
JNeuropathol Exp Neurol •
Kitamura et al
Lack of postoperative irradiation and high Mffi-l index
have previously been reported to be correlated with poor
prognosis of patients with skull base chordomas (5, 9). A
few reports also suggested a trend toward correlation of
incomplete tumor removal with poor prognosis (10, 39).
These findings were confirmed in our study by univariate or
multivariate analyses. Forsyth et al (10) reported that age
«40 years) is strongly associated with longer overall survival
and disease-free survival, but the present study demonstrated
no correlation between age and recurrence. In contrast to some
previous reports that indicated lack of statistical relationship
between sex and prognosis(4,40), however,our study suggested
that female sex might constitute a risk for shorter PFS.
In conclusion, our comprehensive analysis of both
molecular and clinical factors suggests that high Mffi-l index,
loss on lp, gain on lq and 2p, brachyury expression, T gene
CNG, female sex, partial tumor removal, and lack of irradiation are associated with the risk of recurrence or shortening
of PFS in skull base chordomas. Chordomas with these
risk factors should be followed up carefully. Our results
also suggest that brachyury-negative chordomas are biologically distinct from brachyury-positive chordomas and that
T/brachyury might be an appropriate molecular target for
therapy of these neoplasms.
ACKNOWLEDGMENTS
We thank Ms. Naoko Tsuzaki and Ms. Kiyomi Koide
for their technical assistance. We also thank Dr. Masahiko
Maekawa (GSP Laboratory, Inc.) and Dr. Yoshifumi Okada
(Dokkyo Medical University) for their technical advices.
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Control 2001;12:1-11
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Tumor Registry of Japan (1984-2000). Neurol Med Chir (Tokyo) 2009;
49(Suppl):1-96
3. CBTRUS. Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2004-2007, 2011
4. Favre J, Deruaz JP, Uske A, et a1. Skull base chordomas:Presentationof
six cases and review of the literature. J Clin Neurosci 1994;1:7-18
5. Takahashi S, Kawase T, Yoshida K, et al. Skull base chordomas:Efficacy
of surgery followed by carbon ion radiotherapy. Acta Neurochir (Wien)
2009;151:759-69
6. Bugoci DM, GirvigianMR, Chen JC, et al, Photon-based fractionated stereotacticradiotherapy for postoperative trea1ment of skull base chordomas.
Am J Coo Oncol2012 [Epub ahead of print]
7. Geoerger B, Morland B, Ndiaye A, et al. Target-driven exploratorystudy
of imatinib mesylate in children with solid malignanciesby the Innovative Therapies for Children with Cancer (ITCC) European Consortium.
Eur J Cancer 2009;45:2342-51
8. Stacchiotti S, Marrari A, Tamborini E, et al, Response to imatinib plus
sirolimus in advanced chordoma. Ann Oncol 2009;20:1886-94
9. Horbinski C, Oakley GJ, Cieply K, et al, The prognostic value of Ki-67,
p53, epidermal growth factor receptor, Ip36, 9p21, 10q23, and 17p13 in
skull base chordomas. Arch Pathol Lab Med 2010;134:1170-76
10. Forsyth PA, Cascino TL, Shaw EG, et al. Intracranial chordomas: A
clinicopathological and prognostic study of 51 cases. J Neurosurg 1993;
78:741-47
11. Jambhekar NA, Rekhi B, Thorat K, et al. Revisiting chordoma with
brachyury, a "new age" marker: Analysis of a validation study on 51
cases. Arch Pathol Lab Med 2010;134:1181-87
822
Volume 72, Number 9, September 2013
12. Oakley GJ, Fuhrer K, SeethalaRR. Brachyury, SOX-9, and podoplanin,
new markers in the skull base chordoma vs chondrosarcomadifferential:
A tissue microarray-based comparative analysis. Modem Pathol 2008;
21:1461-69
13. Vujovic S, Henderson S, PresneauN, et al. Brachyury, a crucialregulator
of notochordaldevelopment, is a novel biomarkerfor chordomas. J Pathol
2006;209:157-65
14. Yang XR, Ng D, AlcortaDA, et al, T (brachyury)gene duplication confers
major susceptibility to familial chordoma. Nat Genet 2009;41:1176-78
15. Presneau N, Shalaby A, Ye H, et al. Role of the transcription factor T
(brachyury) in the pathogenesis of sporadic chordoma: A genetic and
functional-based study. J PathoI2011;223:327-35
16. Hsu W, Mohyeldin A, Shah SR, et a1. Generation of chordoma cell
line JHC7 and the identificationofbrachyury as a novel molecular target.
J Neurosurg 2011;115:760-69
17. Fernando RI, Litzinger M, Trono P, et al. The T-box transcription factor
brachyury promotes epithelial-mesenchymal transition in human tumor
cells. J Coo Invest 2010;120:533-44
18. Nelson AC, Pillay N, Henderson S, et al. An integrated functional genomics approach identifies the regulatory network directed by brachyury
(T) in chordoma. J Pathol 2012;228:274-85
19. Sasaki H, Yoshida K, Ikeda E, et al. Expression of the neural cell adhesion molecule in astrocytic tumors: An inverse correlation with malignancy. Cancer 1998;82:1921-31
20. Hirose Y, Aldape K, Takahashi M, et al, Tissue microdissection and
degenerate oligonucleotide primed-polymerase chain reaction (DOPPCR) is an effective method to analyze genetic aberrations in invasive
tumors. J Mol Diagn 2001;3:62-67
21. Wolff AC, Hammond ME, Schwartz IN, et a!. American Society of
Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in
breast cancer. J Clin OncoI2007;25:118-45
22. BrandalP, Bjerkehagen B, DanielsenH, et al. Chromosome 7 abnormalities
are common in chordomas. Cancer Genet Cytogenet2005;160:15-21
23. Scheil S, Bruderlein S, Liehr T, et a1. Genome-wide analysis of sixteen
chordomas by comparative genomic hybridization and cytogenetics of
the first human chordomacell line, U-CHI. Genes ChromosomesCancer
2001;32:203-11
24. Le LP, Nielsen GP, Rosenberg AE, et a1. Recurrent chromosomal copy
number alterations in sporadic chordomas. PLoS One 2011;6:eI8846
25. Tallini G, Dorfinan H, Brys P, et al.. Correlation between clinicopathologicalfeaturesand karyotypein 100 cartilaginousand chordoidtumours.
A report from the Chromosomes and Morphology (CHAMP) Collaborative Study Group. J PathoI2002;196:194-203
26. Hallor KH, Staaf J, Jonsson G, et al. Frequent deletion of the CDKN2A
locus in chordoma: Analysis of chromosomal imbalances using array
comparative genomic hybridisation. Br J Cancer 2008;98:434-42
27. Almefty KK, Pravdenkova S, Sawyer J, et a1. Impact of cytogenetic abnormalities on the management of skull base chordomas. J Neurosurg
2009;110:715-24
28. Riva P, Crosti F, Orzan F, et a1. Mapping of candidate region for
chordoma developmentto 1p36.13 by LOH analysis. Int J Cancer 2003;
107:493-97
29. Miozzo M, Dalpra L, Riva P, et a1. A tumor suppressor locus in familial
and sporadic chordoma maps to 1p36. Int J Cancer 2000;87:68-72
30. Longoni M, Orzan F, Stroppi M, et a!. Evaluation of Ip36 markers
and clinical outcome in a skull base chordoma study. Neuro Onco12008;
10:52-60
31. Sawyer JR, Husain M, AI-MeftyO. Identification of isochromosome lq
as a recurring chromosome aberration in skull base chordomas: A new
marker for aggressivetumors? Neurosurg Focus 2001;10:E6
32. Shalaby AA, Presneau N, Idowu BD, et a1. Analysis of the fibroblastic
growth factor receptor-RASIRAFIMEK/ERK-ETS2/brachyury signalling
pathway in chordomas.Mod PathoI2009;22:996-1005
33. Presneau N, Shalaby A, Idowu B, et a1. Potential therapeutic targets for
chordoma: PI3K1AKTrrSClrrSC2/mTOR pathway. Br J Cancer 2009;
100:1406-14
34. Chapiro E, Leporrier N, Radford-Weiss I, et a1. Gain of the short arm of
chromosome 2 (2p) is a frequent recurring chromosome aberration in
untreated chronic lymphocyticleukemia (CLL) at advanced stages. Leuk
Res 2010;34:63-68
© 2013 American Association ofNeuropathologists, Inc.
J Neuropathol Exp Neurol • Volume 72, Number9, September 201 3
35. Jarosova M, Urbankova H, PlachyR, et al. Gain of chromosome 2p in
chronic lymphocytic leukemia: Significant heterogeneity and a new recurrent dicentric rearrangement. Leuk Lymphoma 2010;51 :304-13
36. Beddington RS, Rashbass P, Wilson V. Brachyury-a gene affecting mouse gastrulation and early organogenesis. Development 1992;
116(Suppl):157-65
37. Edwards YH, Putt W, Lekoape KM, et al. The human homolog T of the
mouse T(Brachyury) gene: Gene structure, cDNA sequence, and assignmentto chromosome 6q27.Genome Res 1996;6:226-33
© 2013 American Association ofNeuropathologists, Inc.
Risk Factors in Skull Base Chordoma
38. Tirabosco R, Mangham DC, Rosenberg AE, et al. Brachyury expression
in extra-axial skeletal and soft tissue chordomas: A marker that distinguishes chordoma from mixed tumor/myoepithelioma/parachordoma in
soft tissue. Am J SurgPathol2008;32:572-80
39. al-Mefty 0, BorbaLA. Skullbase chordomas: A management challenge.
J Neurosurg 1997;86:182-89
40. Gao Z, Zhang Q, Kong F, et al. Fascin expression in skull base
chordoma: Correlation with tumor recurrence and dura erosion. Med
Onco12012;29:2438-44
823