Imaging, Diagnosis, Prognosis
Array-Based Comparative Genomic Hybridization Identifies
CDK4 and FOXM1 Alterations as Independent Predictors
of Survival in Malignant Peripheral Nerve Sheath Tumor
Jinsheng Yu1, Hrishikesh Deshmukh1, Jacqueline E. Payton1, Christopher Dunham3,
Bernd W. Scheithauer4, Tarik Tihan5, Richard A. Prayson6, Abhijit Guha7, Julia A. Bridge8,
Rosalie E. Ferner9, Guy M. Lindberg10, Rebecca J. Gutmann2, Ryan J. Emnett2,
Lorena Salavaggione1, David H. Gutmann2, Rakesh Nagarajan1, Mark A. Watson1, and Arie Perry1
Purpose: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with
variable patient survival and few known prognostically relevant genomic biomarkers. To identify survival-
associated genomic biomarkers, we performed high-resolution array-based comparative genomic hybri-
dization (aCGH) on a large set of MPNSTs.
Experimental Design: Candidate gene alterations identified by aCGH in 38 MPNSTs were validated
at the DNA, RNA, and protein levels on these same tumors and an independent set of 87 MPNST
Results: aCGH revealed highly complex copy number alterations, including both previously reported
and completely novel loci. Four regions of copy number gain were associated with poor patient survival.
Candidate genes in these regions include SOX5 (12p12.1), NOL1 and MLF2 (12p13.31), FOXM1 and
FKBP1 (12p13.33), and CDK4 and TSPAN31 (12q14.1). Alterations of these candidate genes and several
others of interest (ERBB2, MYC and TP53) were confirmed by at least 1 complementary methodology,
including DNA and mRNA quantitative real-time PCR, mRNA expression profiling, and tissue microarray-
based fluorescence in situ hybridization and immunohistochemistry. Multivariate analysis showed that
CDK4 gain/amplification and increased FOXM1 protein expression were the most significant independent
predictors for poor survival in MPNST patients (P < 0.05).
Conclusions: Our study provides new and independently confirmed candidate genes that could
serve as genomic biomarkers for overall survival in MPNST patients. Clin Cancer Res; 17(7); 1924–34.
Malignant peripheral nerve sheath tumors (MPNST) are
highly aggressive and frequently lethal Schwann cell-
derived neoplasms associated with poor survival (1–5).
Roughly half of MPNSTs develop sporadically and the
other half arise in the setting of neurofibromatosis type
patients is reportedly half of that for sporadic cases (21%
vs. 42%; ref 5). However, other studies suggest that a
diagnosis of NF1 does not affect survival (6). Clinicopatho-
logic factors, such as tumor grade and anatomic site report-
edly influence MPNST patient outcome, although the
clinical course remains highly variable and difficult to
predict in individual patients.
Traditional cytogenetic studies have reported that
MPNSTs have remarkably complex karyotypes with a wide
spectrum of chromosomal alterations including transloca-
it difficult to discern which of these alterations are biolo-
gically relevant (7–9). Unfortunately, no single alteration
Authors' Affiliation: Departments of1Pathology and Immunology, and
2Neurology, Washington University School of Medicine, Saint Louis, Mis-
souri;3Division of Anatomic Pathology, Children's and Women's Health
Centre of B.C., Vancouver, British Columbia, Canada;
Anatomic Pathology, Mayo Clinic, Rochester, Minnesota;5Department of
Pathology, University of California in San Francisco, San Francisco,
California;6Department of Anatomic Pathology, Cleveland Clinic Founda-
tion, Cleveland, Ohio;7Division of Neurosurgery, University Health Net-
work, Toronto, Ontario, Canada;8Department of Orthopaedic Surgery,
University of Nebraska Medical Center, Omaha, Nebraska;9Department of
Clinical Neuroscience, King's College London, London, United Kingdom;
and10Pathologist, Caris Diagnostics, Dallas, Texas
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/)
Current address for A. Perry: Department of Pathology, Division of Neu-
ropathology, University of California, San Francisco, San Francisco, CA.
Corresponding Author: Arie Perry, Department of Pathology, Division of
Neuropathology, University of California, San Francisco, 505 Parnassus
Avenue, #M551, Box #0102, San Francisco, CA 94143. Phone: 415-476-
5236 or 415-476-4961; Fax: 415-476-7963. E-mail: Arie.Perry@ucsf.edu.
?2011 American Association for Cancer Research.
Clin Cancer Res; 17(7) April 1, 2011
has consistently proven to be diagnostically or prognosti-
on chromosome 17 (where the NF1 gene is located) are
expected, given that MPNSTs are highly over-represented
among NF1 patients. However, NF1 losses are not always
encountered (8–10), suggesting that other tumorigenic
pathways also exist. A major limitation of traditional cyto-
genetic studies, however, has been their limited resolution
of ?10 Mb. This has complicated the discovery of specific,
recurring genetic alterations involving small regions.
High-resolution genome-wide DNA microarray analyses
have identified many novel tumor-specific microdeletions
and amplifications in leukemia, lung, and ovarian cancers
(11–13). To date, a similar approach applied to MPNSTs
has been limited to few studies with either less compre-
hensive or lower resolution array platforms (?1 Mb; refs
14–16). In the present study, we have analyzed a larger
cohort of MPNSTs utilizing both Affymetrix and Nimble-
(aCGH) platforms (0.25–2.5 kb of resolution with whole
genome coverage) to identify recurrent DNA copy number
alterations (CNA) that are associated with reduced patient
survival, independent of traditional clinical and patholo-
gical parameters in patients with this extremely heteroge-
of candidate genes with CNAs which may prove clinically
useful as prognostic biomarkers for patients with MPNST.
Materials and Methods
Patients and specimens
A cohort of 38 patients with histologically confirmed
MPNST was included in this study for microarray analysis.
Among the 38 patients, 23 (60%) individuals carried a
previous diagnosis of NF1 and 15 (40%) were sporadic
cases. At the time of last follow-up, 14 (37%) cases had
documented metastases, whereas 16 (42%) had no evi-
dence of metastatic disease. Recurrence data were unavail-
able for 8 of the cases. The majority (33/38) of cases
subjected to genomic analysis was primary tumors,
although 5 cases of recurrent tumors were also included.
A full accounting of clinical and pathological data is
provided in Supplemental Table T1. DNAs and RNAs
were extracted from frozen tumors, using commercial
reagents (QIAamp DNA Mini Kit, Qiagen; Trizol reagent,
Invitrogen) and following manufacturers’ standard pro-
tocols. These same specimens were previously utilized
for gene expression profiling (17). For biomarker valida-
tion studies, another independent set of 121 formalin-
fixed paraffin-embedded MPNST tumors represented in
a tissue microarray (TMA) was utilized; 87 of these
patients had clinical annotation and follow-up data
(18). Of these 87 annotated cases, 25 (29%) were NF1-
associated and 62 (71%) were sporadic MPNSTs. A total
of 68 primary, 12 locally recurrent, and 7 metastatic
tumors were represented. The majority of the tumors
(78/87) were high grade and 9/87 were classified as low
grade. Supplemental Table T2 provides additional data
regarding the validation set. All tissue specimens and
clinical data from these patients were collected and uti-
lized in accordance with Institutional Review Board–
Array-based comparative genome hybridization
The Affymetrix Genome-Wide 500K SNP Mapping
array set was employed to detect genomic alterations
in the set of 38 MPNSTs. For aCGH analysis, relative
copy number values of the MPNSTs were generated using
a reference set of 37 nonmalignant DNA samples derived
from 22 males and 15 females. To identify meaningful
CNAs, 2 DNA copy number analysis tools, Partek Geno-
mics Suite (PARTEK, www.partek.com) and Copy Num-
ber Analyzer for GeneChip (CNAG, www.genome.umin.
jp; ref 19) were applied to the aCGH data. CNAs identi-
fied with each of the 2 tools were compared with identify
recurrent regions with minimal overlap. Physical posi-
tions of CNAs were annotated according to human gen-
ome build 35 (www.ncbi.nlm.nih.gov). All CNAs were
filtered against the human normal copy number varia-
tion (CNV) database (http://projects.tcag.ca/variation)
prior to further analysis. CNAs identified on the 500K
SNP platform were also confirmed in a subset of 8
patients where patient-matched tumor and germline
DNAs were available, using a dual-color high-density
NimbleGen 135K aCGH platform (see Supplemental
Text for details).
Quantitative real-time PCR
SYBR Green chemistry was utilized in quantitative real-
time PCR (qRT-PCR) assays; qRT-PCR primer sequences
are listed in Supplemental Table T3. The qRT-PCR was
Malignant peripheral nerve sheath tumor (MPNST) is
an aggressive malignancy with considerable biological
variability, a variableand often lethalclinical course, and
limited response to current therapeutic approaches. A
has been a lack of available prognostic or predictive
biomarkers. Through initial identification of several sur-
vival-associated copy number alterations, followed by
confirmatory assays and a comprehensive multivariate
analysis, our present study demonstrates that CDK4
sion are significant independent predictors for poor sur-
vival in MPNST patients. If confirmed in additional
prospective studies, these biomarkers may provide clini-
cally useful information for managing patients with this
aggressive malignancy. Interestingly, a previous report
has demonstrated that the multi-kinase inhibitor sorafe-
nib can inhibit MPNST growth, suggesting the potential
of CDK4 as a therapeutic target and also as a potential
predictive biomarker of response to sorafenib therapy.
Genomic Biomarkers and Survival in MPNST
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