Paired tumor and normal whole genome sequencing of metastatic olfactory neuroblastoma.

Abstract

Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.
Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.
This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.

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Paired Tumor and Normal Whole Genome Sequencing of
Metastatic Olfactory Neuroblastoma
Glen J. Weiss1,2*, Winnie S. Liang3, Tyler Izatt4, Shilpi Arora2, Irene Cherni2, Robert N. Raju5,
Galen Hostetter6, Ahmet Kurdoglu3, Alexis Christoforides3, Shripad Sinari3, Angela S. Baker6,
Raghu Metpally7, Waibhav D. Tembe7, Lori Phillips3, Daniel D. Von Hoff1,8., David W. Craig4.,
John D. Carpten6.
1Virginia G. Piper Cancer Center Clinical Trials at Scottsdale Healthcare (VGPCC), Scottsdale, Arizona, United States of America, 2Cancer and Cell Biology Division, The
Translational Genomics Research Institute, Phoenix, Arizona, United States of America, 3Collaborative Sequencing Center, The Translational Genomics Research Institute,
Phoenix, Arizona, United States of America, 4Neurogenomics Division, The Translational Genomics Research Institute, Phoenix, Arizona, United States of America,
5Innovation Center, Kettering, Ohio, United States of America, 6Integrated Cancer Genomics Division, The Translational Genomics Research Institute, Phoenix, Arizona,
United States of America, 7Collaborative Bioinformatics Center, The Translational Genomics Research Institute, Phoenix, Arizona, United States of America, 8Clinical
Translational Research Division, The Translational Genomics Research Institute, Phoenix, Arizona, United States of America
Abstract
Background: Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We
performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to
identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.
Methodology/Principal Findings: Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole
blood, followed by WGS at .30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to
confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside
coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by
Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were
present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR,
MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a
result of post-treatment effects.
Conclusions/Significance: This work provides insight into the evolution of ONB cancer cells and provides a window into the
more complex factors, including tumor clonality and multiple driver mutations.
Citation: Weiss GJ, Liang WS, Izatt T, Arora S, Cherni I, et al. (2012) Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory
Neuroblastoma. PLoS ONE 7(5): e37029. doi:10.1371/journal.pone.0037029
Editor: Hassan Ashktorab, Howard University, United States of America
Received October 31, 2011; Accepted April 11, 2012; Published May 23, 2012
Copyright: ? 2012 Weiss et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: National Foundation for Cancer Research www.nfcr.org TGen Foundation www.tgenfoundation.org National Institutes of Health grants
#1S10RR25056-01 and #1S10RR023390-01 www.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gweiss@tgen.org
. These authors contributed equally to this work.
Introduction
Previously called esthesioneuroblastoma, olfactory neuroblasto-
ma (ONB) is a rare cancer comprising 2% of all sinonasal tract
tumors with an incidence of 0.4 cases per million [1]. ONB is
thought to arise from sensory neuroepithelial olfactory cells
typically found in the upper portion of the naval cavity [1]. These
tumors do not have a gender predilection and can occur at any
age, but have a bimodal age distribution in the 2ndand 6thdecades
of life [1]. The most common presenting symptoms include
unilateral nasal obstruction (70%), and epistaxis (50%). Anosmia is
not a common complaint (5%) [1]. ONB is a malignant tumor and
,25% of the patients develop cervical lymph node metastasis [2].
Based on pathology, distinguishing features of ONB include
nesting, neurofibrillary stroma and presence of stippled nuclei. Its
distinct immunoprofile includes loss of keratin expression,
immunopositivity for neuroendocrine markers, and S100 positive
cells surrounding the nests of tumor cells. Despite all these
distinguishing features, the wide variability in these tumors can
lead to difficulty in diagnosis [3]. Surgery and radiation with or
without chemotherapy are considered the standard of care for
non-distant metastatic disease based primarily on retrospective
series [4]. While no standard chemotherapy exists for ONB,
cisplatin and etoposide or doxorubicin, or vincristine with an
alkylating agent are most commonly administered [5]. However,
after such treatment ONB often recurs [6].
Due to the rarity of this disease, most of the published literature
on ONB includes case reports or retrospective analysis of ONB
patients to predict treatment outcome. There have been very few
studies on the molecular characterization of ONB. Expression of
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Bcl-2, Trk-A and B, Grp78 and several other markers has been
analyzed by immunohistochemistry by different groups [7,8].
Array comparative genomic hybridization (aCGH) has revealed
multiple chromosomal aberrations in this tumor type [9]. The
study by Guled et al. analyzed 13 ONB samples and revealed copy
number changes including gains at 7q11.22-q21.11, 9p13.3, 13q,
20p/q, and Xp/q, and losses at 2q31.1, 2q33.3, 2q37.1, 6q16.3,
6q21.33, 6q22.1, 22q11.23, 22q12.1, and Xp/q [9]. In addition,
the Hedgehog signaling pathway has been posited to be crucial for
ONB development [6]. A study by Koschny et al. showed that
primary ONB cells are TRAIL (TNF related apoptosis inducing
ligand) resistant but are sensitized to TRAIL-induced apoptosis by
the proteasome inhibitor bortezomib. This sensitizing effect
involves several regulators of the TRAIL signaling pathway. Both
these anti-cancer agents are already in clinical use but their effect
on ONB patients remain to be evaluated [10]. Sequencing
analyses have identified new genes and pathways that have not
been previously linked to human cancer [11,12]. Apart from these
studies there is little information on the genomic alterations or
changes in cellular signaling in ONB patients. Thus, so far there
has been no study to identify mutation profiles of these rare
cancers in order to identify new therapeutic targets for treating
these patients.
It is universally accepted that somatic alterations (i.e., point
mutations, small insertions and deletions, rearrangements, gains
and losses) occur at the DNA level in cancer. These somatic
events can drive tumorigenesis, metastatic progression, and/or
drug resistance[13].More
alterations are intimately tied to companion targeted therapeu-
tics. Technological advances have been rapid and in 2007, the
first genome sequence of a single individual was deciphered in
only 2 months at a cost of less than $2 million [14].
Researchers from the Wellcome Trust in UK published the
entire genome of two cancer cell lines [15,16]. These studies of
a malignant melanoma and a lung cancer line, respectively,
revealed for the first time almost all of the mutations in the
genomes of these two cancers [15,16]. In order to take full
advantage of these technological advances, we have applied
such capabilities in the clinic and translated them to the
management ofdiseasein individual
physicians have few choices when formulating a treatment plan
for a patient with advanced cancer especially in case of rare
cancers, as there is usually very little published literature about
these diseases. Thus, we are far from understanding the genetics
and disease progression in these diseases. As demonstrated,
whole genome sequencing (WGS) technologies now provide us
with platforms to interrogate entire human genomes at a
fraction of the time and cost compared to more traditional
sequencing technologies. These technologies, for the first time,
offer us the ability to survey the global somatic landscape of
cancer. It is now possible with WGS to re-sequence, analyze,
and compare the matched tumor and normal genes of an
individual patient. With these paradigm shifts in technologic
capabilities, we present the results of the first paired tumor and
normal whole genome sequences of metastatic ONB.
importantly,specificsomatic
patients.Currently,
Methods
Ethics Statement
The study was approved by the Western Institutional Review
Board (WIRB) and was conducted in accordance with the 1996
Declaration of Helsinki. This was a pilot study entitled, ‘‘An
Ancillary Pilot Trial Using Whole Genome Tumor Sequencing in
Patients with Advanced Refractory Cancer’’ (WIRBH Protocol
#20101288)(NCT01443390). Informed consent was obtained
from the patient with olfactory neuroblastoma, including written
consent for publication of the clinical details and images.
Eligibility Criteria
Patients of age $18 that provided signed informed consent,
with relapsed or refractory solid tumors, who progressed on at
least one systemic therapy for advanced disease and willing to
undergo a biopsy or surgical procedure to obtain tissue, which
may not be a part of the patient’s routine care for their malignancy
were eligible for the study. Other eligibility criteria included:
Karnofsky performance status (PS) $80%, life expectancy .3
months, baseline laboratory data indicating acceptable bone
marrow reserve, liver, and renal function. Patients were allowed
to participate on another clinical trial involving treatment prior to
or during participation on this study. Main exclusion criteria
included: symptomatic central nervous system (CNS) metastasis,
untreated CNS metastases, known active infections requiring
intravenous antimicrobial therapy, known HIV, HBV or HCV
infection requiring antiviral therapy, pregnant or breast feeding
women, or inaccessible tumor for biopsy. While additional patients
were recruited under this protocol to perform WGS of both their
tumor DNA and germline DNA, no other patient had a diagnosis
of ONB or head and neck cancer.
Case Report
A 29-year-old man with metastatic ONB presented to
Virginia G. Piper Cancer Center Clinical Trials at Scottsdale
Healthcare for participation in a pilot study to apply WGS on a
fresh biopsy of one of his metastatic lesions to determine if
identification of somatic perturbations might be useful for
downstream therapy. The initial diagnosis of ONB was made
after expert pathologic review at a major academic center. The
tumor consisted of nests of closely packed cells with small to
medium sized nuclei and scant cytoplasm. Within the nests,
single cell and occasional necrosis was present. The tumor had
features of epithelial differentiation confirmed by pankeratin
staining, which highlights the clusters and single cells within the
nests with strong cytoplasmic staining. The rest of the cells stain
strongly for synaptophysin supporting the diagnosis of high
grade ONB. The tumor was negative for chromogranin,
neurofilament, CD45, CD20, S100, HMB45, and Melan A.
Subsequently collected specimens also underwent confirmatory
pathology review for the ONB diagnosis.
Sample Assessment
Samples were received for tumor confirmation, analyte (DNA)
extraction and processing for downstream WGS experiments. All
tumor samples were obtained under IRB approved protocol, were
preserved as fresh frozen and reference DNA was obtained from
peripheral blood mononuclear cells. Samples from the patient with
ONB were collected at SHC (Scottsdale Healthcare, Scottsdale,
AZ) under radiographic guidance, flash frozen in liquid nitrogen
and transported to TGen (The Translational Genomics Research
Institute, Phoenix, AZ) on dry ice and stored at 280uC until
sample processing. Direct visualization of samples collected was
obtained by two-ink frozen quality control (QC) procedure to
estimate tumor content and extent of tissue heterogeneity by a
board-certified pathologist (GH). All tumor samples used had
greater than 50% tumor content. Digital image files of all QC
tissues were scanned using Aperio GL scanner and image files
were stored on secure web-based viewing by Spectrum Plus
(Aperio, Inc).
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Genomic DNA Isolation
Fresh frozen tissue.
guided 18-gauge needle biopsy was disrupted and homogenized
in Buffer RLT plus, Qiagen AllPrep DNA/RNA Mini Kit, using
the Bullet BlenderTM, Next Advance. Specifically, approximately
7 mg of tissue was transferred to a microcentrifuge tube containing
600 ml of Buffer RLT plus, and 9 mg of 1.6 mm stainless steel
beads. The tissue was homogenized in the Bullet Blender at room
temperature at Speed 10 for 5 minutes. The sample was
centrifuged at full speed and the lysate was transferred to the
Qiagen AllPrep DNA spin column. Genomic DNA purification
was conducted as directed by the AllPrep DNA/RNA Mini
Handbook, Qiagen. DNA was quantified by Nanodrop spectro-
photometer for appropriate dilution and quality was accessed
using 260/280 and 260/230 absorbance ratios.
Whole blood.
Genomic DNA was isolated from frozen blood
using the Qiagen QiaAmp DNA Blood Midi Kit. Specifically,
blood (12 ml) that was previously frozen and stored at 280uC was
allowed to equilibrate to room temperature. Two ml of blood was
transferred to conical tubes and treated with protease. Each 2 ml
aliquot was then applied to Qiagen QiaAmp Midi columns and
genomic DNA was purified as directed by the Qiagen QiaAmp
DNA Blood Midi/Maxi Handbook. DNA was quantified using the
Nanodrop spectrophotometer and quality was accessed from 260/
280 and 260/230 absorbance ratios.
Whole genome library preparation.
from each sample (control and tumor) was used for library
preparation. Samples were prepared for sequencing using
proprietary methods. In summary, samples were fragmented
using the Covaris S2 system (part#4387833) to a target fragment
size of 300 to 350 base pairs (bp). Fragmentation was verified by
running samples on a 2% Tris Acetate EDTA (TAE) gel.
Overhangs in the fragmented samples were then repaired to form
blunt ends using T4 DNA polymerase and Klenow (New England
Biolabs; NebNext DNA Sample Prep Master Mix Set I; catalog #
E6040L), and products were cleaned using Agencourt Ampure
magnetic beads (Beckman Coulter Genomics; catalog # A29153).
Adenine bases were next ligated onto the blunted fragments using
Klenow exo (NebNext DNA Sample Prep Master Mix Set I), and
A-tailed products were cleaned using Ampure magnetic beads.
Products were next quantified using Quant-iT Picogreen dsDNA
reagent (Invitrogen; catalog # P11496) in triplicate with a 0 ng/
mL to 200 ng/mL standard. To prepare for adaptor ligation,
samples were vacuum dried to the appropriate volume to allow for
a 10:1 adaptor to DNA molar ratio. Diluted paired end Illumina
adapters were then ligated onto the A-tailed products using DNA
ligase (NebNext DNA Sample Prep Master Mix Set I). Following
ligation, samples were run on a 3% TAE gel at 120V for 2.5 hours
to separate ligated products. X-tracta gel extractors (USA
Scientific; catalog # 5454-0100) were used to select ligation
products at 300 bp and 350 bp for each sample. Ligated products
were isolated from these gel punches using Freeze ‘N Squeeze
DNA Gel Extraction Spin Columns (Bio-rad; catalog # 732-
6166), and cleaned using Ampure magnetic beads. 2X Phusion
High-Fidelity PCR Master Mix (Finnzymes; catalog # F-531L)
was used to perform PCR in quadruplicate (10 uL ligation/
reaction) to enrich for these products. Enriched PCR products
were run on a 2% TAE gel and were selected using xtracta gel
extractors. PCR products were purified from gel punches using
Freeze ‘N Squeeze DNA Gel Extraction Spin Columns. Extracted
products were purified using Ampure magnetic beads and
quantified using Agilent’s High Sensitivity DNA chip (catalog #
5067-4626) on the Agilent 2100 Bioanalyzer (catalog #
G2939AA).
Tissue collected from ultrasound-
3 mg of genomic DNA
Paired end next generation sequencing.
normal libraries were prepared for paired end sequencing.
Samples were denatured using 2N NaOH and diluted with
Illumina HT2 buffer. Clusters were generated on all flowcells
using Illumina’s cBot and HiSeq Paired End Cluster Generation
Kits. Flowcells were sequenced on Illumina’s HiSeq 2000 using
Illumina’s HiSeq SBS Sequencing Kit for two paired end 100 bp
sequencing runs per flowcell.
Data analysis.
The Illumina HiSeq 2000 generated raw
sequence data in the form of .bcl files. These data were converted
to .qseq files using Illumina’s BCL Converter tool, and resulting
.qseq files were used to generate .fastq files for downstream
analysis. Fastq files were validated to evaluate the distribution of
quality scores and to ensure that quality scores do not drastically
drop over each read. Validated fastq files were aligned to the
human reference genome (build 36) using the Burrows-Wheeler
Alignment (bwa) tool [17], which uses the Burrows-Wheeler
Transform (BWT) algorithm. Following alignment, generated .sai
files were used to create .sam (sequence alignment map) files by
converting suffix array coordinates to chromosomal coordinates
[17]. Resulting .sam files were input into SAMtools [18] to create
binary sequence (.bam) files. PCR duplicates were flagged for
removal using Picard (http://picard.sourceforge.net), and base
quality scores were recalibrated using GATK (Genome Analysis
Toolkit) [19]. Mutation analysis was performed to identify single
nucleotide polymorphisms (SNPs), insertion/deletions (indels), and
copy number variants. Results from all mutation analyses are
summarized in a Circos plot [20].
Single nucleotide variant (SNV) identification.
calling was performed using SolSNP (http://sourceforge.net/
projects/solsnp/files/SolSNP-1.01/) and Mutation Walker, a tool
developed in house and that incorporates variant discovery tools
from GATK. SNPs that were called using both tools were
compiled for further examination. Two sets of thresholds, strict
and lenient, were enabled to reduce the false negative rate. Data
from each of these two sets were visually examined for false
positives to create a final filtered list of true SNVs, which were
annotated with GENCODE using an internal annotation script.
SolSNP is an individual sample mutation detector implemented
in Java. The algorithm is based on modified Kolmogorov-Smirnov
like statistics, which incorporates base quality scores. The
algorithm is non-parametric and makes no assumptions on the
nature of the data. It compares the discrete sampled distribution,
the pileup on each strand, to the expected distributions (according
to ploidy). In the case of a diploid genome, both strands need to
provide evidence for the variation. Zero quality bases are trimmed
off the pileup before comparison. SolSNP is a standalone program
that can be run from the command line and is general enough to
work with any next generation sequencing data with high
coverage. While making somatic calls, SolSNP’s high quality
genotype call is made for all callable loci of the normal sample. To
reduce false negatives, variant loci in tumor samples are called
with the Variant Consensus mode. Variant loci in tumor samples
that exhibit a high quality homozygous reference genotype in the
normal sample are considered as somatic. To call somatic variants,
SolSNP is augmented by a Python script.
Insertion/deletion identification.
formed using GATK and a somatic indel detection tool
developed in house. For detecting somatic indels we employed
a two-step strategy. In the first step, we removed reads whose
insert size laid outside the interval (50,500) from the tumor bam
files. GATK was then used to generate a list of potential small
indels from this bam. A customized Perl script, which uses the
Bio-SamTools library from BioPerl [21], takes these indel
Tumor and
SNP
Indel calling was per-
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positions and for each of the indels, looks at the region in the
normal sample that is 5 bases upstream from the start of the
indel and 5 bases downstream from the end of the indel. An
indel was determined to be somatic only if there was no indel
detected in the region under consideration.
Copy number analysis.
Copy number variants were iden-
tified by an analysis of differential clone coverage. In this
assessment, a single fragment includes the two sequenced paired
ends and the unsequenced interlying region as being covered by
one clone. Unmapped reads and reads lying within 2 standard
deviations outside of the insert distance are not included. Relative
copy number is determined as the log2 difference between the
normal and tumor normalized coverage, where normalization is
the mean coverage across a 2 kb window divided by the genome-
wide mode coverage.
Validation of Mutations Identified by WGS
DNA extraction from
embedded (FFPE) tissue sections.
tained under an approved IRB protocol. H&E sections were
prepared from each patient block and areas of tumor cells were
marked. A 12 mm section was then cut for each patient block and
respective H&E sections were overlaid to identify areas of tumor
cell enrichment. Next, the tissue sections were deparaffinized and
areas of tumor cells were scraped and DNA extractions were
performed using RecoverAll kit from Ambion (Invitrogen,
Carlsbad, CA) according to manufacturer’s protocol.
Sanger sequencing to confirm mutations.
primers were designed for the genomic sequences for each of the
mutated genes chosen for further validation. Primers were
designed such that PCR products ranged from 200–350 bp.
Primer sequences for the seven genes chosen for the validation of
mutations are included in Table S1. M13 sequences were included
in the forward and reverse primers specific to each gene as a back-
up for sequencing reactions. DNA was extracted from each of the
samples as described above and 10 ng DNA was used for each
specific PCR reaction. Platinum Taq high fidelity DNA polymer-
ase (Invitrogen Inc., Carlsbad, CA) was used for the PCR and the
reactions were performed according to manufacturer’s protocols.
PCR was run for 35 cycles of: Denaturation: 94uC for 20 sec,
Annealing: 58.5uC for 30 sec and elongation: 68uC for 30 sec.
Upon completion, PCR products were purified using QIAquick
PCR purification kit (Qiagen Inc., Valencia, CA) and sent for
PCR sequencing to Arizona State University DNA sequencing
Core facility. All the reactions were forward and reverse sequenced
using specific forward and reverse primers. In a few cases, M13
primers were also utilized for the sequencing reactions.
theformalin-fixed,
FFPE blocks were ob-
paraffin-
Specific PCR
Results
Case Report
A 29-year-old man with metastatic ONB presented to Virginia
G. Piper Cancer Center Clinical Trials at Scottsdale Healthcare
for participation in a pilot study to apply WGS on a fresh biopsy of
one of his metastatic lesions to determine if identification of
somatic perturbations might be useful for downstream therapy.
Prior to the cancer diagnosis, his past medical history was
significant for obesity. Pertinent past environmental exposure
history included tobacco chewing for five years, and chemical or
fume exposures in the work place (tool/dye shop). In early 2008,
he presented with symptoms of sinus congestion, sore throat,
headache, and unexplained vomiting. Evaluation and imaging at
another institution revealed a 4.2 cm mass in the nasal cavity
extending into the ethmoid and frontal sinuses. Biopsy confirmed a
diagnosis of ONB. He underwent surgery involving both head and
neck and neurosurgery consisting of an anterior craniofacial
resection, sphenoidectomy, total ethmoidectomy, and transnasal
craniotomy in June 2008. He then received radiation therapy
66.6 Gy over 37 fractions from June to September 2008 with
concurrent carboplatin and vincristine. After radiotherapy,
consolidation carboplatin and vincristine chemotherapy was
delivered through March 2009.
In June 2009, he underwent a dacryocystorhinostomy for
epiphora, and showed no evidence of cancer. Subsequently, he had
development of several local regional recurrences including nasal
bridge recurrence inDecember 2009.Heunderwent extensiveskull
base surgery at the end of January 2010 including neck nodal
dissection.Oneof21lymphnodeshadmicroscopicmetastaticdisease
and he had tumor that was extending to the ethmoid region to the
orbital wall. After undergoing plastic reconstruction with a right
forearm free flap, cranioplasty and skin grafting, radiation therapy
(totaldoseof54 Gy)wasdeliveredtotherightneckfromMarch2010
to May 2010. A right cheek metastasis measuring 363 cm was
confirmedbyexcisionalbiopsyinJuly2010.Heunderwentasurgical
debridement of the forehead flap in August 2010. In October 2010,
tumorrecurrenceontheleftridgewasconfirmedbyabiopsyandwas
visible on the bilateral nasal and ocular canthi, and suspected to
involvethe right parotid.
At presentation to our clinic, he was on a liquid diet but could
swallow pills. His main complaint was xerostomia, and he had
anosmia, moderate fatigue, and frontal pressure headaches
requiring opiate pain medication. His Karnofsky performance
status was 80, and he had right-sided exophthalmos limiting his
ability to fully close the right eyelid. Pertinent physical exam
findings included several visible areas of metastases in the nasal
ridge and orbital ridge, and a palpable hard, fixed mass in the
right parotid region (Figure S1A photos). Radiographic imaging
was conducted at our institution and revealed multiple foci of
tumor (Figures S1B-H CT/MRI images).
Whole Genome Sequencing
Signed informed consent was obtained and after confirming
eligibility, the patient underwent a biopsy in the right parotid
region for tumor DNA collection and a venous blood draw for
normal germline DNA. WGS was performed after DNA
isolation. We generated 463.5GB of total sequencing data with
an average Q30 percentage of 81.3% for a total of 379.6GB of
Q30 data. We achieved average coverages of 71X for the
normal sample and 68X for the tumor. A total of 2,173,398
SNPs were found in the germline with 87% existing in dbSNP
and a 2.1 transition/transversion rate. A total of 5,789 SNVs
were found across the genome. Of these, sixty-two somatic non-
synonymous or missense SNVs and 5 coding indels were
identified inside coding regions–these events each cause a non-
synonymous change in the DNA sequence as shown in the
Circos plot [20] (Figure 1, 2 and Table S2). These indels were a
base deletion of CYP4A22 (1:47384338 del T), a seven base
insertionin
KCNA10
(1:110861798
insertion of 1 bp in OBSCN (1:22657241 ins T), a 1 base
deletion in ARID4B (1:233411716 Del A), and 7 base deletion in
CCDC120 (X:48811750 del TCGTAGC). A total of 119 genes
were found to be somatically lost resulting from a near complete
single copy deletion of chromosome 18, focal deletions at 5q15,
6 p25.1, 7p15.3, 7p21.3, 11q24.2, 19p12, and 21q.1. By
comparison, a total of 45 genes were found to be gained or
amplified resulting from amplification of 8p, focal events at
5p15.33, 7p13, 8q24.3, 9q22.31, 9q34.3, 16q22.1, and 16q24.3.
A total of 4/104 coding SNVs were also found in 1000
insGAGCAAC),an
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Genomes database of germline variation, consistent with a less
than 5% false positive rate of SNVs actually being germline
events that were poorly covered. Frequently mutated genes
based on the Cosmic database (Sanger) include FGFR1 [22],
FANCC, NOTCH1 [23], and CBFA2T3 were all amplified and
JAZF1 was deleted. Other key genes include amplifications of
RXRA, NSMAF, and ASPH [24]. Deletions of other key genes
include ETS1 [25,26,27], CCNH, and coagulation factor XIII A1
(F13A1).
Validation of Mutations Identified by WGS
Seven mutated genes namely, MAP4K2, SIN3B, TAOK2, KDR,
TP53, MYC, and NLRC4 were chosen to validate the presence of
specific gene mutations as shown in Table 1. This selection was
based on clinical relevance and previously published literature on
the target genes and their association with carcinogenesis. SIFT
results classified all the mutations except NLRC4, as damaging.
Similar results were seen after Polyphen protein analysis, which
classified the mutation in NLRC4 as benign, while all the others
were classified as damaging. For validation, primers were designed
Figure 1. Circos plot for WGS results for ONB. This figure depicts the genomic location in the outer ring and chromosomal copy number in the
inner ring. The SNVs and indels are marked on the outer ring in their respective genomic locations. In the inner ring, copy gains are shown in red,
while copy losses are shown in green. No interchromosomal translocations were observed by assessing counts of anomalous read pairs between
specific regions of the genomes, noting that the use of shorter-paired end sequencing may limit our ability to detect these events with this analysis.
doi:10.1371/journal.pone.0037029.g001
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for specific gene amplification (Table S1) and the PCR products
were Sanger sequenced. Sanger sequencing confirmed mutations
in all seven genes (Figures S2A-D) in the patient sample used for
WGS. Next, we examined these seven validated mutations in
previously collected archival FFPE samples from this ONB patient
(Table 2). In the metastatic FFPE samples of the right parotid
collected several months prior to WGS, all seven mutations were
present. Interestingly, in the original surgical resection specimens
(prior to evidence of metastatic disease), mutations in KDR, MYC,
SIN3B, and NLRC4 genes were not present, while TP53, MAP4K2
and TAOK2 mutations were present in the original surgical
specimen as well.
Discussion
ONB is a rare cancer of the sinonasal tract and data on its
molecular characterization is limited. We performed WGS on
paired normal and tumor DNA from a patient with metastatic
ONB in an attempt to survey the somatic alterations that might be
drivers of tumorigenesis and/or metastatic progression in this
disease. After validation of seven selected SNVs in the sequenced
tumor sample, we analyzed archival metastatic and primary
resection FFPE samples available from the same patient. We found
that specific mutations in KDR, MYC, SIN3B, and NLRC4 appear
only in the metastatic setting, while mutations in TP53, TAOK2
and MAP4K2 were also present in the previous samples (original
biopsies of the ONB tumor). There was a mutation in codon 176
in the p53 gene, which results in a residue change from a cysteine
to a phenylalanine. Majority of mutations in p53 occur in the core
domain that contains the sequence-specific DNA binding activity
of the p53 protein, and the mutations result in loss of DNA
binding activity. The core domain structure consists of two loops
(L2 and L3) and the LSH motif and this particular mutation
(codon 176) is in the L2 loop [28]. The L2 loop, although not
directly involved in DNA binding, is involved in extensive
interactions with the L3 loop for binding together zinc atom
(Cys176 on the L2 loop is a ligand for the zinc) [29]. In another
study, tumors from 26 ONB samples microscopically more closely
resembled paragangliomas, and aberrant expression of TP53 was
noted in 62% of cases [30]. Absence of TP53 mutations in 14
ONB cases with hyper-expression of wild-type TP53 was found in
four metastatic/recurrent cases. One study suggested that TP53
point mutations did not play an important role in the initial
development of ONB as wild-type TP53 hyper-expression may
lead to local aggressive behavior and a tendency for recurrence
[31]. Immunohistochemical analysis of one ONB case revealed
that 10% of the tumor stained positive for TP53 protein and
vascular endothelial growth factor (VEGF) [32]. This mutation has
been previously associated with several other cancer types
including oral cancers and Ewing’s sarcoma [29,33]. In the study
involving oral cancers, both the patients with this particular
mutation died of the disease within 12 months [29].
Figure 2. Differences between the germline and somatic sequences. Details the statistics for the germline SNPs and somatic SNVs.
doi:10.1371/journal.pone.0037029.g002
Table 1. Single nucleotide variations in the seven genes chosen for validation by Sanger sequencing.
GENE IDCHR Position Coding
Codon
Position
Reference
Codon
Reference
amino acid
Mutated
Codon
Mutated
Amino acidType SIFT results
Polyphen
results
MAP4K2 1164313974 CDS 761AGCSATCI nsSNP DamagingPossibly
damaging
TAOK21629898286 CDS 204 GGCGGACD nsSNP ToleratedPossibly
damaging
TP5317 7519128CDS176 TGCCTTCFnsSNP Damaging Possibly
damaging
SIN3B19 16834366 CDS421AAGKATGMnsSNPDamagingPossibly
damaging
NLRC4232329712CDS242 AGGRAAGKnsSNPTolerated Benign
KDR455671617 CDS351CCTPCTTLnsSNPDamagingPossibly
damaging
MYC8128820394 CDS235CCCPCTCL nsSNP Damaging
LC*
Possibly
damaging
Key: *Low confidence, Mutated Codon column-somatic mutation depicted in bold face.
doi:10.1371/journal.pone.0037029.t001
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Similarly mutations in MYC and KDR, though not as common
as p53 mutations, have been previously described and associated
with cancer [34,35]. Castaneda et al have shown N-MYC and
MYC expression in a primary ONB sample by northern blot
analysis [36]. Several of these genes/their protein products are
known to interact/regulate other genes on the list and play
important roles in cancer cell signal transduction. For example,
MYC binds to SIN3B promoter directly [37]. Similarly, p53 binds
to NLRC4 promoter and activates its expression in a cell-type-
specific manner [38]. Thus, it would be interesting to study these
interactions in vitro in cell line model systems in which these genes
were mutated.
Our work has identified and validated seven mutations in an
ONB patient. In addition, we tested these mutations in additional
samples from the same patient. This work provides insight into the
evolution of cancer cells and provides a window into the more
complex factors at play here, including tumor clonality and
multiple driver mutations. Based on these data it is clear that the
tumor displays additional mutations. Normal course of disease
progression could be responsible for their acquisition. Alternative-
ly, additional mutations could have arisen in response to the
primary radiation and/or carboplatin/vincristine chemotherapy
as a mechanism of resistance. These mutations may have been
present in the subclones of the primary tumor and selected for by
dynamic tumor microenvironment. Lastly, as tumors from only
one individual were sequenced we do not know which of the SNVs
reported here are driver mutations as opposed to passenger
mutations occurring by chance. All these hypotheses need to be
tested in optimal in vitro model systems. Additionally, the mutated
target genes and their cellular signaling mechanisms indicate
aberrations in DNA repair mechanisms, which may be related to
ONB progression in this patient. A limitation of this study is the
lack of additional metastastic ONB tumors to assess by WGS.
With the reduction in cost, improvement in speed of analysis and
with more complete understanding of complex genetic alterations,
we anticipate that WGS will be applied in the clinic more
frequently to common and rare cancers and will pave the way to
personalized medicine.
Supporting Information
Figure S1
(PPTX)
Imagesofmetastaticolfactoryneuroblastoma.
Figure S1A
mass in the right parotid region that was subsequently biopsied for
tumor whole genome sequencing.
(TIF)
Photograph of metastatic olfactory neuroblastoma
Figure S1B
recurrence of the olfactory neuroblastoma. Arrow points to the
heterogeneous mass.
(TIF)
CT axial image depicting extent of local disease
Figure S1C and S1D
and FLAIR sequence (1D) MRI axial images depicting extent of
local disease recurrence of the olfactory neuroblastoma. Arrow
points to the heterogeneous mass.
(TIF)
Spin echo T1 weighted with contrast (1C)
Figure S1E and S1F
and spin echo fast scan (1F) MRI coronal images depicting extent
of local disease recurrence of the olfactory neuroblastoma. Arrows
point to the heterogeneous mass.
(TIF)
Spin echo T1 weighted pre-contrast (1E)
Figure S1G and S1H
spin echo fast scan image (1H) depicting metastasis to the right
parotid region. Arrow points to the mass that was biopsied for
tumor whole genome sequencing.
() TIF
CT axial image (1G) and MRI coronal
Figure S2
harboring SNVs. DNA alignments and sequencing electrophe-
rograms depicting specific SNVs in KDR (Figures S2A and S2B)
and MYC (Figures S2C and S2D) genes. Arrows in Figures S2B
and S2D point to the mutated residue in the electropherograms.
Electropherogram shown for KDR (Figure S2B) is for the
sequencing reaction of the complementary strand.
(PDF)
Genomic regions of KDR and MYC genes
Table S1
sequencing of the mutated regions. Key: *The sequences
in bold are the M13 forward and reverse primer sequences added
to our specific primers to aid in sequencing reactions.
(XLS)
Primers used for PCR amplification and
Table S2
(XLS)
Summary of mutated genes, N=67.
Table 2. Validation of mutations in previously collected archival FFPE samples from the ONB patient.
Sample DateSite examined MYCKDRTAOK2 MAP4K2SIN3BTP53NLRC4Comments
6/2/08Left nasal massCCC AGG
GACATCAAG TTCAGG At diagnosis, mutations in
TAOK2/MAP4K2/TP53
Right nasal mass CCCAGG
GAC ATCAAGTTCAGG
6/12/08Frontal tumor CCCAGG
GACATCAAGTTCAGGResection samples do not
have new mutations
Nasal tumorCCCN/A
GACATCAAGTTC AGG
7/26/10Right cheek
CTCAAGGACATCATGTTCAAGMetastatic samples show
evolution of new
mutations in MYC/KDR/
SIN3B/NLRC4
Right cheek
CTCAAGGACATCATGTTC AAG
11/3/10Right cheek (WGS) CTC AAGGACATCATGTTCAAG
CCCAGGGGCAGCAAGTGCAGGReference codons
Key: Validated mutations compared to reference codon depicted in bold italics.
doi:10.1371/journal.pone.0037029.t002
Olfactory Neuroblastoma Whole Genome Sequencing
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Page 8

Acknowledgments
We thank the patients and their families, and the clinical staff including
Vickie Marsh, and laboratory staff including Carlos Lorenzo, Aprill
Watanabe, and Guy Raz who provided technical assistance.
Author Contributions
Conceived and designed the experiments: GJW SA DDVH DWC JDC.
Performed the experiments: WSL TI SA IC GH AK AC SS ASB RM LP.
Analyzed the data: GJW WSL TI SA IC RM WDT DDVH DWC JDC.
Contributed reagents/materials/analysis tools: GJW WSL RNR DWC
JDC. Wrote the paper: GJW WSL TI SA IC DDVH DWC JDC.
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