Recurrent IDH2 R172X mutations in sinonasal undifferentiated carcinoma

Abstract

Sinonasal undifferentiated carcinoma is a rare and aggressive malignancy. Sinonasal undifferentiated carcinoma has long been considered a diagnosis of exclusion; to date, the molecular pathogenetic basis for sinonasal undifferentiated carcinoma is unknown. To identify potential oncogenic drivers in sinonasal undifferentiated carcinoma, targeted next-generation sequencing of 300 cancer-related genes was performed on 11 cases of sinonasal undifferentiated carcinoma. We identified IDH2 R172X mutations in 55% of sinonasal undifferentiated carcinomas including R172S, R172T, and R172M. Multispecific mutant IDH1/2 immunohistochemistry was performed and identified mutant-specific protein expression in all cases with available tissue: 3/3 sinonasal undifferentiated carcinomas with R172 mutations were positive and 4/4 wild-type cases were negative. Review of sequencing data for our institutional head and neck cohorts (n=412) confirmed the absence of IDH-activating mutations in other tumor types. Alterations in the IDH2-wild-type sinonasal undifferentiated carcinomas included SMARCA4 loss-of-function with confirmed loss of immunohistochemical expression, NOTCH1 gain-of-function, and TET2 loss-of-function. We demonstrate that the majority of histologically defined sinonasal undifferentiated carcinomas are characterized by IDH2 R172X mutations and overexpression of mutant protein. IDH2 R172X mutations are specific to sinonasal undifferentiated carcinoma among carcinomas of the head and neck, confirming this tumor type as a distinct clinicopathologic entity. These findings have significant implications for diagnosis and therapy with IDH inhibitors for patients with this rare and poorly understood tumor.Modern Pathology advance online publication, 13 January 2017; doi:10.1038/modpathol.2016.239.

Full text

Recurrent IDH2 R172X mutations in sinonasal
undifferentiated carcinoma
Vickie Y Jo1, Nicole G Chau2, Jason L Hornick1, Jeffrey F Krane1and Lynette M Sholl1,3
1Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA;
2Department of Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA,
USA and 3Center for Advanced Molecular Diagnostics, Brigham and Women’s Hospital and Harvard Medical
School, Boston, MA, USA
Sinonasal undifferentiated carcinoma is a rare and aggressive malignancy. Sinonasal undifferentiated carcinoma
has long been considered a diagnosis of exclusion; to date, the molecular pathogenetic basis for sinonasal
undifferentiated carcinoma is unknown. To identify potential oncogenic drivers in sinonasal undifferentiated
carcinoma, targeted next-generation sequencing of 300 cancer-related genes was performed on 11 cases of
sinonasal undifferentiated carcinoma. We identified IDH2 R172X mutations in 55% of sinonasal undifferentiated
carcinomas including R172S, R172T, and R172M. Multispecific mutant IDH1/2 immunohistochemistry was
performed and identified mutant-specific protein expression in all cases with available tissue: 3/3 sinonasal
undifferentiated carcinomas with R172 mutations were positive and 4/4 wild-type cases were negative. Review of
sequencing data for our institutional head and neck cohorts (n=412) confirmed the absence of IDH-activating
mutations in other tumor types. Alterations in the IDH2-wild-type sinonasal undifferentiated carcinomas included
SMARCA4 loss-of-function with confirmed loss of immunohistochemical expression, NOTCH1 gain-of-function,
and TET2 loss-of-function. We demonstrate that the majority of histologically defined sinonasal undifferentiated
carcinomas are characterized by IDH2 R172X mutations and overexpression of mutant protein. IDH2 R172X
mutations are specific to sinonasal undifferentiated carcinoma among carcinomas of the head and neck,
confirming this tumor type as a distinct clinicopathologic entity. These findings have significant implications for
diagnosis and therapy with IDH inhibitors for patients with this rare and poorly understood tumor.
Modern Pathology advance online publication, 13 January 2017; doi:10.1038/modpathol.2016.239
Sinonasal undifferentiated carcinoma is a poorly
characterized, rare, and aggressive epithelial
malignancy of the sinonasal tract. Sinonasal
undifferentiated carcinoma most commonly afflicts
older adults, although a wide age range has been
observed.1Sinonasal undifferentiated carcinoma
presents as a fast growing, locally destructive and
invasive tumor, often extending into the cranial
vault.2Mortality is high, with an overall 2-year
survival rate o50% despite the multimodal ther-
apy.3,4 As sinonasal undifferentiated carcinoma was
first described in 1986,5few insights have been made
into its pathogenesis, and no current standardized
treatment regimen exists. Tumors originally consid-
ered to represent sinonasal undifferentiated carci-
noma are likely heterogeneous, as genomically
defined carcinomas have been subsequently identified
within this ‘undifferentiated’group, including NUT-
midline carcinoma,6,7 nasopharyngeal carcinoma,8,9
HPV-related carcinomas,10 and SMARCB1 (INI1)-
deficient carcinoma.11,12
Uniform pathologic diagnostic criteria for
sinonasal undifferentiated carcinoma are not well
established; this tumor type has long been consid-
ered a diagnosis of exclusion once epithelial
differentiation is confirmed and other entities
have been excluded. Tumors express keratins
and EMA but no other differentiation markers with
rare exceptions, including focal staining for
synaptophysin, chromogranin, S-100, and p63/
p40.5,9,13–15 Thus far, no recurrent genetic alterations
have been identified in sinonasal undifferentiated
carcinoma16 and no specific diagnostic markers
exist.
Our aim was to evaluate a cohort of sinonasal
undifferentiated carcinoma using large panel-
targeted next-generation sequencing to identify
recurrent genetic alterations, in an effort to better
define this poorly understood and lethal tumor.
Correspondence: Dr VY Jo, MD, Department of Pathology, Brigham
and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA.
E-mail: vjo@partners.org
Received 17 October 2016; revised 12 December 2016; accepted 13
December 2016; published online 13 January 2017
Modern Pathology (2017), 1–10
© 2017 USCAP, Inc All rights reserved 0893-3952/17 $32.00 1
www.modernpathology.org

Materials and methods
Tumor Samples
Eleven cases of sinonasal undifferentiated carcinoma
diagnosed between 2007 and 2016 were retrospec-
tively identified in the surgical pathology archives.
Hematoxylin and eosin- and immunohistochemi-
cally stained slides were reviewed by two specialists
in head and neck pathology (VYJ and JFK) for
diagnostic confirmation, excluding known mimics
when possible. For example, NUT-midline carcino-
mas, SMARCB1 (INI1)-deficient carcinomas, and
nasopharyngeal undifferentiated carcinomas (posi-
tive by in situ hybridization for Epstein–Barr
virus-encoded RNA; EBER) were excluded, as were
tumors that showed strong and diffuse p63 or p40
immunoreactivity. Morphologic features were
reviewed blinded to immunohistochemical and
sequencing results. This study was performed with
approval by the Institutional Review Board at
Brigham and Women's Hospital and Dana Farber
Cancer Institute (Boston, MA, USA).
Sequencing Analysis
Next-generation sequencing was performed at Brig-
ham and Women's Hospital. The OncoPanel assay
surveys DNA sequences of all coding exons of 300
cancer-related genes and 113 introns across 35 genes
for rearrangement detection using massively parallel
sequencing (see Supplementary Table 1 for a
complete list of targeted genes). DNA was extracted
from formalin-fixed paraffin-embedded tumor sam-
ples using QIAamp DNA Mini Kit (Qiagen, Valencia,
CA, USA), and then sheared into fragments of 250 bp
average size with a sonicator (Covaris, Woburn, MA,
USA). TruSeq Sample Preparation Kit (Illumina, San
Diego, CA, USA) was used to construct DNA
libraries. Pools of single-stranded RNA probes were
designed and synthesized as part of the Agilent
SureSelect Hybrid Capture Kit (Agilent, Santa Clara,
CA, USA) and used to bait DNA of interest from the
sample library. Selected DNA was quantified, nor-
malized, and pooled, and then sequenced with an
Illumina HiSeq 2500 sequencer. Sequence data were
demultiplexed and aligned using the Burrows-
Wheeler Aligner software tool. The MuTect and
GATK software was used to detect single-nucleotide
variants and small insertion-deletions (indels),17,18
and copy number variations were detected using
RobustCNV, a tool that is based on the calculation of
log2ratio of read counts of individual specimens
against a panel of normal tissues. BreakMer analysis
was performed to detect large structural variations as
described previously.19 Any calls present at 40.1%
in the Exome Variant Server (http://evs.gs.washing
ton.edu/EVS/) were automatically filtered, with a
rescue for any variants also present in Cosmic
(http://cancer.sanger.ac.uk/cosmic) two or more
times. All single-nucleotide variants, copy number
variations, and translocation calls were manually
reviewed and annotated for pathogenetic signifi-
cance (LMS). Alterations were classified on a 4-tier
scheme according to clinical relevance: Tier 1,
actionable target using a Food and Drug
Administration-approved therapy; Tier 2, prognostic
implications or clinical trial drug target; Tier 3,
inferred biological relevance; and Tier 4, variant of
unknown significance.
Sequencing data from an institutional cohort of
head and neck tumors (n= 412; including three
sinonasal undifferentiated carcinomas from this
cohort) assayed by OncoPanel under a consented
cancer sequencing program (PROFILE, DFCI IRB nos
11–104)20 were reviewed using an institutional
instance of cBioPortal.21
Immunohistochemistry and In Situ Hybridization
IDH2 immunohistochemistry was performed on
formalin-fixed paraffin-embedded whole sections
from seven cases with available tumor tissue follow-
ing pressure cooker antigen retrieval (pH 6.1; Target
Retrieval Solution; Dako, Carpinteria, CA, USA)
using a mouse monoclonal antibody for IDH1/2
mutant R132/172 (clone MsMab-1; 1:100 dilution;
Millipore, Darmstadt, Germany) with Novolink
Detection System (Leica, Buffalo Grove, IL, USA).
A colorectal adenocarcinoma with known IDH2
R172S mutation was used as a positive control.
Immunohistochemistry results were scored blinded
to the IDH2 mutational status. Positive staining was
characterized by granular cytoplasmic staining in
tumor cells. The other antibodies and conditions for
the immunohistochemical studies performed for this
cohort are detailed in Table 1. The Envision Plus
detection System (Dako, Carpinteria, CA, USA) was
used for all other antibodies besides IDH1/2.
In situ hybridization for EBER was performed
on a Ventana Benchmark XT autostainer (Ventana,
Tucson, AZ, USA) using an 1 DNP probe (Ventana;
cat. no. 760-1209A) and ISH iView Blue Plus anti-
DNP Detection System (Ventana).
Statistics
Survival time calculation was performed using Stata/
IC 12.1 (StataCorp LP, College Station, TX, USA).
Results
Clinical and Pathologic Features
The clinical characteristics of the cohort are sum-
marized in Table 2. Three cases were diagnosed in
pathologic consultation only and had limited clinical
data available. The cohort consisted of six men and
five women, aged 41–75 years (median 64), and
included six neversmokers. Sampled tumor sites
were nasal (n= 5), nasopharynx (n= 1), maxillary
IDH2 R172X in sinonasal undifferentiated carcinoma
2VY Jo et al
Modern Pathology (2017) 00, 1 –10

sinus (n= 1), ethmoid sinus (n= 2), sphenoid sinus
(n= 1), and pleural effusion with metastasis from a
maxillary sinus primary (n=1).
All sinonasal undifferentiated carcinomas showed
sheet-like and nested growth of medium-to-large
cells with hyperchromatic and vesicular nuclei,
prominent nucleoli, and scant-to-moderate palely
eosinophilic cytoplasm (Figure 1). All cases had
necrosis and brisk mitotic activity (median count of
18.5 per 10 high-power fields (range, 5–47)). Lym-
phovascular and perineural invasion were frequent.
No evidence of squamous differentiation (keratiniza-
tion) or associated surface squamous dysplasia was
observed; duct or gland formation and rhabdoid
cytoplasmic inclusions were absent in all cases. All
tumors showed strong and diffuse keratin expression
(11/11). Rare reactivity was seen for p63/p40 (1/10),
synaptophysin (2/10), chromogranin (3/9), and S-100
(2/9). Negative stains included desmin (0/7), NUT
(0/8), and EBER (0/6); expression of SMARCB1 (INI1)
was retained (7/7), and SMARCA4 (BRG1) expres-
sion was lost in 1/7 (see below). Table 3 summarizes
the pathologic features and immunohistochemical
results for the cohort.
Next-Generation Sequencing Results
The median overall sequencing coverage per case
was 384 reads (range, 126–610), and the median
percentage of sequences achieving 430-fold cover-
age was 99% (range, 97–99%). The median number
of single-nucleotide variants per tumor using this
targeted panel was 3.1 per megabase (range 1.5–4.1
per Mb). Figure 2 summarizes the key genomic
findings in the 11 cases of sinonasal undifferentiated
carcinoma. A complete list of identified single-
nucleotide variants and structural variants is avail-
able in Supplementary Table 2.
IDH2 mutations at the known hotspot R172 were
identified in 6 of 11 (55%) sinonasal undifferentiated
carcinomas, including R172S (n= 4), R172T (n= 1),
and R172M (n= 1) (Figure 1). Review of sequencing
data for 409 other head and neck malignancies
confirmed the absence of IDH-activating mutations
in all other tumor types (see Supplementary Table 3),
including 6 NUT-midline carcinomas, 4 nasophar-
yngeal carcinomas, 5 neuroendocrine carcinomas, 3
olfactory neuroblastomas, and 319 squamous cell
carcinomas (including 11 primary sinonasal tumors).
Five sinonasal undifferentiated carcinomas were
IDH2-wild type. One case had a NOTCH1 PEST
domain insertion mutation leading to premature
truncation (Case 7); this is a documented mechanism
of NOTCH pathway activation in other tumor
types.22 ASMARCA4 splice site variant and two
concomitant APC loss-of-function variants (Figure 2
and Supplementary Table 2) were detected in Case 8;
SMARCA4 loss of expression was confirmed by
immunohistochemistry (Figure 3). A BAP1 splice
site mutation along with single-copy deletion of
BAP1 was observed in Case 9. Case 11 had a TET2
exon 3 frameshift mutation.
Diverse concomitant oncogenic alterations,
including in PIK3CA,MTOR,SOX2, and SOX9 were
also identified in the IDH2-mutated tumors. Loss-of-
function alterations likely contributing to cell cycle
dysregulation, including in TP53,RB1, and
CDKN2A, were identified in three IDH2-mutated
cases and one IDH2-wild-type case (Figure 2 and
Supplementary Table 2). Loss-of-function alterations
of multiple genes involved in epigenetic regulation
(including ARID1A,ARID2,CREBBP,KMT2A,
KMT2D,SETD2,TET2) were identified in 4/6
IDH2-mutated tumors and 8/11 overall (Figure 2
and Supplementary Table 2). In contrast to acute
myeloid leukemia,23 TET2 mutations were identified
both together with, and exclusive of, IDH2 mutations
(Figure 2).
Copy number variant profiles were available in
nine cases; copy number variant data could not be
interpreted for Cases 10 and 11 because of low tumor
content. Recurrent low copy gains were identified at
1q21–1q44 (5/6 IDH2-mutated sinonasal undifferen-
tiated carcinomas; 6/9 overall), 8q13.1–8q24.3 (4/6
IDH2-mutated sinonasal undifferentiated carcino-
mas; 6/9 overall), and 17q23.2–17q24.3 (4/6 IDH2-
mutated sinonasal undifferentiated carcinomas; 5/9
Table 1 Antibodies and conditions used for immunohistochemistry
Antibody Source Clone Dilution Pretreatment
Pankeratin Dako (Carpinteria, CA, USA) MNF116 1:700 10 min protease digestion
p63 Neomarkers (Fremont, CA, USA) 4A4 1:600 Citrate buffer, pressure cooker
p40 EMD Millipore (Darmstadt, Germany) Polyclonal 1:1000 Citrate buffer, pressure cooker
Synaptophysin Leica (Buffalo Grove, IL, USA) 27612 1:50 None
Chromogranin Thermo Scientific (Waltham, MA, USA) LK2H10 1:4000 Citrate buffer, pressure cooker
S-100 protein Dako Polyclonal 1:1000 None
Desmin Sigma (St Louis, MO, USA) DE-U-10 1:5000 Citrate buffer, pressure cooker
NUT Cell Signaling (Danvers, MA, USA) C52B1 1;200 Citrate buffer, pressure cooker
SMARCB1/INI1 BD Biosciences (San Jose, CA, USA) Mo25 1:200 Citrate buffer, pressure cooker
IDH1/2 132/172 Millipore MsMab-1 1:100 Citrate buffer, pressure cooker
SMARCA4/BRG1 Abcam (Cambridge, MA, USA) ERP3912 1:50 Citrate buffer, pressure cooker
Abbreviation: EBER, Epstein–Barr virus-encoded mRNA.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
VY Jo et al 3

overall), including SOX9 amplification in two IDH2-
mutated cases. Copy number variations at the
chromosomal arm level are summarized in Figure 4.
IDH1/2 Immunohistochemistry
IDH1/2 immunohistochemistry was performed in
seven samples with available remaining tissue.
Three of three sinonasal undifferentiated carcinomas
with R172 mutations showed cytoplasmic staining
for IDH1/2. Two of these tumors had R172S and
showed moderate-to-strong multifocal-to-diffuse
reactivity (Figure 1). One tumor harbored R172M
and showed weak-to-moderate multifocal staining
(Figure 1). No staining was observed in 4/4 IDH2-
wild-type sinonasal undifferentiated carcinomas
(Figure 5).
Clinical and Pathologic Features of Patients with
IDH2-mutated and -Wild-Type Sinonasal
Undifferentiated Carcinomas
Comparisons of the clinical features of the patients
with IDH2-mutated and IDH2-wild-type tumors are
limited by the small cohort size, thus no statistically
robust conclusions can be drawn; however, the
patients with IDH2-mutated tumors were younger
(median 57 years) versus IDH2-wild-type (median 71
years). All patients presented with similar stages of
disease; eight patients with known clinical data
underwent radiation and chemotherapy, and surgi-
cal resection was performed for 1/4 patients with
IDH2-mutated tumors and 3/4 IDH2-wild-type
patients. Overall median survival for the cohort is
23 months; small numbers and limited follow-up
time preclude comparison of outcomes in IDH2-
mutated and -wild-type cohorts.
No morphologic or immunophenotypic differ-
ences were detected between IDH2-mutated and
wild-type tumors.
Discussion
Sinonasal undifferentiated carcinoma is a rare
aggressive neoplasm appearing as an undifferen-
tiated or poorly differentiated epithelial malignancy.
We performed an analysis of sinonasal undifferen-
tiated carcinoma using targeted next-generation
sequencing of 300 oncogenes and tumor suppressors.
Our study demonstrates that sinonasal undifferen-
tiated carcinomas, when defined based on morphol-
ogy and the presence of strong keratin expression per
traditional criteria, are genomically heterogeneous
but enriched for activating IDH2 mutations. Notably,
all IDH2 mutations occur at the known hotspot R172,
the identification of which creates a novel,
genomically defined subset of sinonasal carcinomas.
Examination of the genomic sequences of over 400
other tumor types including morphologic mimics
Table 2 Clinical characteristics of 11 patients with sinonasal
undifferentiated carcinoma
Characteristics Number
Sex
M6
F5
Patient age, median (range), years 64 (41–75)
Race
Caucasian/non-hispanic 7
Other 1
Unknown 3
Smoking history
Former 2
Never 6
Unknown 3
Alcohol use
Yes 4
No 4
Unknown 3
Occupational/carcinogen exposure
Yes 1
No 7
Unknown 3
Radiation exposure
Yes 1
No 7
Unknown 3
Primary tumor site
Nasal cavity 5
Ethmoid sinus 2
Maxillary sinus 2
Sphenoid sinus 1
Nasopharynx 1
Involvement of multiple sinuses 8
Extension into orbit and cranial structures 8
Duration of tumor-related symptoms preceding diagnosis
o1 month 1
1–3 months 5
46 months 2
Unknown 3
AJCC stage at initial diagnosis
T4aN0M0 (stage IVA) 2
T4bN0M0 (stage IVB) 4
T4bN0M1 (stage IVC) 1
T4aN2bM1 (stage IVC) 1
Unknown 3
Treatment
Surgery followed by adjuvant chemoradiation 4
Induction chemotherapy followed by chemoradiation 3
Palliative chemotherapy alone 1
Unknown 3
Clinical course (n=8)
Follow-up, median no. of months (range) 21 (1–33)
Locoregional recurrence 2
Distant metastases 5
Bone 2
Lung/pleural 2
Liver 2
CNS 3
Overall disease-specific survival 23 months
Abbreviations: AJCC, American Joint Committee on Cancer; CNS,
central nervous system; F, female; M, male.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
4VY Jo et al

indicates that IDH2 mutations are specific to sino-
nasal undifferentiated carcinoma in the differential
diagnosis with other head and neck carcinomas.
The identification of IDH2 mutations has signifi-
cant potential implications for management of
sinonasal undifferentiated carcinoma. Isocitrate
dehydrogenase catalyzes the oxidative decarboxyla-
tion of isocitrate to α-ketoglutarate (α-KG). Mutations
in IDH1 at R132 and IDH2 at R172 alter the activity
of the IDH1 and IDH2 enzymes, catalyzing the
reduction of α-KG to 2-hydroxyglutarate (2HG).
2HG is an oncometabolite that is structurally
analogous to α-KG and can interfere with histone
demethylation mediated by a family of α-KG-depen-
dent enzymes.24 The oncogenic role of mutant IDH1
and IDH2 has been demonstrated in several human
malignancies marked by a hypermethylated pheno-
type and blocked cellular differentiation, including
subsets of acute myeloid leukemia and glioma, as
well as chondrosarcomas.25–27 Based on evidence
that mutant IDH1/2 inhibitors can induce differentia-
tion in acute myeloid leukemia and gliomas,28,29
IDH1/2 mutations are currently being investigated
as biomarkers of response to IDH inhibitors. The
identification of loss-of-function mutations in the
α-KG-dependent DNA demethylase TET2, including
in an IDH2-wild-type tumor, suggests that epigenetic
dysregulation (eg, hypermethylation or impaired
demethylation) is a common feature in sinonasal
undifferentiated carcinoma.23 Further studies are
needed to confirm this hypothesis.
Although some recurrent copy number alterations
were observed, including low gains on chromosomes
1, 8, and 17, no pattern of copy gains and losses
clearly distinguished IDH2-mutant and wild-type
tumors. There was variability in the number of copy
number variants even within the IDH2-mutated
group; some tumors had relatively few gains and
Figure 1 Sinonasal undifferentiated carcinomas with IDH2 R172X mutations. Case 6: (a) Right nasal mass biopsy, hematoxylin and eosin
(H&E) stain; (b)IDH2 c.516G4C (p.R172S) (Integrated Genome Viewer, Broad Institute, Cambridge, MA, USA); (c) Immunohistochemistry
for IDH1/2 R132/172 shows strong and diffuse granular cytoplasmic staining. Case 3: (d) Right ethmoid mass biopsy, H&E; (e)IDH2
c.515G4T (p.R172M) (Integrated Genome Viewer); (f) immunohistochemistry for IDH1/2 R132/172 shows weak-to-moderate multifocal
granular cytoplasmic staining. Case 5: (g) nasal mass biopsy, H&E; (h)IDH2 c.516G4C (p.R172S) (Integrated Genome Viewer); (i)
immunohistochemistry for IDH1/2 R132/172 shows moderate to strong and diffuse granular cytoplasmic staining. All photomicrographs at
x400 magnification.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
VY Jo et al 5

losses (Case 4), whereas multiple high-level ampli-
fications (including SOX2,ETV6,SOX9) and a
homozygous deletion (NF2) were observed in
Case 6. The presence of copy number heterogeneity
across the IDH2-mutated sinonasal undifferentiated
carcinomas suggests the presence of differing levels
of genomic instability, which may have implications
for differential prognosis and response to therapy
even within this genetically defined set of tumors.
The identification of IDH2 R172X mutations also
has important diagnostic implications for this tumor
type, formerly considered a diagnosis of exclusion.
Indeed, a subset of cases can be identified by
immunohistochemistry using a multispecific anti-
body for mutant IDH1/2 R132/172. However, this
antibody has been shown to lack sensitivity for
R172T;30 therefore, at this time-sequencing meth-
odologies may be necessary to detect the range of
IDH2 variants as well as other potentially targetable
alterations in histologically similar tumors.
The differential diagnosis of sinonasal undiffer-
entiated carcinoma can be challenging and requires
ancillary testing to exclude morphologic mimics.
Previous series and diagnoses of sinonasal undiffer-
entiated carcinoma likely encompass a heteroge-
neous group of tumors that has been narrowed down
over the past several decades with the increasing
recognition of pathogenetically defined tumor types
and the development of diagnostic biomarkers.
Immunohistochemistry, in situ hybridization, and
molecular testing can now identify many specific
entities within keratin-positive poorly differentiated
or undifferentiated malignancies in the sinonasal
tract. The differential diagnosis is broad, and
includes squamous cell carcinoma and its variants
(including HPV-associated carcinoma, nasopharyn-
geal carcinoma, and NUT-midline carcinoma) and
SMARCB1 (INI1)-deficient sinonasal carcinoma, as
well as neuroendocrine carcinoma, olfactory neuro-
blastoma, and salivary neoplasms such as the solid
variant of adenoid cystic carcinoma. Nasopharyngeal
carcinoma is typically associated with a dense
Table 3 Pathologic characteristics of 11 cases of sinonasal undifferentiated carcinoma
Features
Case
12 3 4567891011
Tumor size (cm) 4.8 6.9 6.4 N/A 4.8 N/A 4.6 2.0 4.2 N/A N/A
Necrosis present Y Y Y Y Y Y Y Y Y Y Y
Mitotic count (per 10 HPF/2.5 mm2)25 10 12 47 28 23 14 5ND
aNDaNDa
IHC/ISH
Keratin +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
p63/p40 −− − −−+−N/A −−−
Synaptophysin −− +−−−−+−−N/A
Chromogranin + −+ N/A −+−−−−N/A
S-100 −− +−−−+ N/A −−N/A
EBER ISH −−N/A −N/A −N/A N/A N/A −−
NUT −− − −−−N/A N/A −−N/A
SMARCB1 (INI1)bN/A +++ +++ N/A +++ +++ +++ N/A +++ +++ N/A
SMARCA4 (BRG1)bN/A N/A +++ N/A +++ +++ +++ −+++ +++ N/A
IDH2 status R172S R172T R172M R172S R172S R172S WT WT WT WT WT
IDH1/2 R132/172 N/A N/A +++ N/A +++ +++ −N/A −−−
Abbreviations: HPF, high-power field; IHC, immunohistochemistry; ISH, in situ hybridization; N, no; WT, wild type; Y, yes.
aND due to insufficient tissue in biopsy for 10 consecutive HPFs.
bPositive staining indicates intact protein (ie, normal).
Figure 2 Single-nucleotide variants, structural variants, and copy
number alterations as determined by targeted next-generation
sequencing for selected genes in 11 cases of sinonasal undiffer-
entiated carcinoma.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
6VY Jo et al

lymphocytic infiltrate, and appears as an undiffer-
entiated carcinoma with sparse (if any) histologic
features of squamous differentiation; p63/p40 can
confirm squamous differentiation and the diagnosis
can be confirmed by in situ hybridization to detect
Epstein–Barr virus-encoded mRNA.8,9 NUT-midline
carcinoma, defined by rearrangements of the NUT
gene (encoded on 15q14), frequently arises in the
sinonasal tract; while appearing primarily as a
poorly differentiated or undifferentiated carcinoma,
foci of abrupt keratinization may be present and
tumor cells are positive for p63/p40. CD34 is positive
in less than half of all cases. NUT immunostain will
show a punctate nuclear staining pattern;7,31 FISH
can also confirm NUT gene rearrangement.
SMARCB1 (INI1)-deficient sinonasal carcinoma typi-
cally has an infiltrative nested or trabecular growth
pattern within a fibrous stroma, with tumor cells
having round nuclei, prominent nucleoli, and vary-
ing basaloid or epithelioid appearances, and rhab-
doid morphology at least focally present.11,12,32
SMARCB1 (INI1)-deficient sinonasal carcinoma
shows variable p63/p40 staining (up to 44%) and
occasional p16 reactivity, and demonstrates consis-
tent loss of expression for SMARCB1 (INI1) second-
ary to SMARCB1 deletion.11,12
Although our cohort of sinonasal undifferentiated
carcinomas was enriched for IDH2 mutants, it is
notable that other significant potential driver events
were identified in several of the IDH2-wild-type
tumors. Inactivating mutations located within the
NOTCH1 PEST regulatory domain lead to increased
intracellular Notch expression and may predict
therapeutic benefit with γ-secretase inhibitors.33
Interestingly, similar NOTCH1 mutations have been
reported in the solid variant of adenoid cystic
carcinoma,34 a potential morphologic mimic of
sinonasal undifferentiated carcinoma. A MYB-NFIB
Figure 3 Sinonasal undifferentiated carcinoma with SMARCA4 splice variant (c.3774+1G4A) (Case 8). (a) Sphenoid sinus mass,
hematoxylin and eosin (H&E) stain. (b) Immunohistochemical loss of expression of SMARCA4 protein. All photomicrographs at x400
magnification.
Figure 4 Copy number alterations as determined by targeted next-
generation sequencing in 11 cases of sinonasal undifferentiated
carcinoma. Dotted lines indicate centromere position, and solid
lines demarcate chromosomes. Light red = low copy gain; dark
red = amplification (estimated ≥6 copies); light blue = single-copy
deletion; dark blue = homozygous deletion. Tumor content in
Cases 10 and 11 was too low for copy number change detection.
Cases 1–6 represent IDH2-mutated tumors.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
VY Jo et al 7

rearrangement was not detected in the NOTCH1-
mutated tumor reported here. SMARCA4 deficiency
as a driver event in tumorigenesis has been reported
in a variety of undifferentiated carcinomas and
sarcomas arising at other sites;35–37 to our knowl-
edge, this phenomenon has not yet been described in
head and neck malignancies, but is not unexpected
given the recent recognition of SMARCB1 (INI1)-
deficient sinonasal carcinoma,11,12,32 with which the
SMARCA4-deficient sinonasal carcinoma might be
classified.
While the specific association with IDH2 muta-
tions argues that sinonasal undifferentiated carci-
noma deserves recognition as an independent dia-
gnostic entity, it remains to be determined whether
mutant IDH2 sinonasal undifferentiated carcinoma
represents a distinct clinicopathologic entity.
The detection of other driver mutations (NOTCH1
gain-of-function, SMARCA4 loss-of-function, and
TET2 loss-of-function) suggests that there may be
other potential genomically defined subsets within
histologically defined sinonasal undifferentiated
carcinomas. Limitations of our study include a small
cohort and short follow-up time. Given the potential
role for targeted inhibitors, pathologists and clin-
icians should consider sequencing to determine
IDH2 status for patients with a diagnosis of sinonasal
undifferentiated carcinoma. Multi-institutional stu-
dies, including prospective identification of IDH2
mutations and use of basket trials to test the activity
of mutant IDH inhibitors in patients with this very
rare disease, will be required to determine the true
clinical implications of this finding in sinonasal
undifferentiated carcinoma. In conclusion, the pre-
sence of recurrent IDH2 R172X mutations in sinona-
sal undifferentiated carcinoma begins to redefine
Figure 5 Negative IDH1/2 R132/172 immunohistochemistry in IDH2-wild-type sinonasal undifferentiated carcinoma. Case 7: (a)
Sinonasal undifferentiated carcinoma, right maxillary sinus, hematoxylin and eosin stain; (b) negative staining by IDH1/2 R132/172
immunohistochemistry. Case 9: (c) Sinonasal undifferentiated carcinoma, left ethmoid sinus, hematoxylin and eosin stain; (d) negative
staining by IDH1/2 R132/172 immunohistochemistry. Case 10: (e) Sinonasal undifferentiated carcinoma, nasopharynx, hematoxylin and
eosin stain; (f) negative staining by IDH1/2 R132/172 immunohistochemistry. Case 11: (g) Metastatic sinonasal undifferentiated in a cell
block from a pleural effusion (from a right maxillary sinus primary), hematoxylin and eosin stain. (h) Negative staining by IDH1/2
R132/172 immunohistochemistry. All photomicrographs at x400 magnification.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
8VY Jo et al

this ‘diagnosis of exclusion’and opens up genomi-
cally driven clinical trial opportunities for patients
with this rare and aggressive form of carcinoma.
Acknowledgments
This work was funded by the Department of
Pathology, Brigham and Women's Hospital.
Disclosure/conflict of interest
The authors declare no conflict of interest.
References
1 Frierson HF. Sinonasal undifferentiated carcinoma. In:
Barnes L, Eveson JW, Reichart P, et al. (eds). WHO
Classification of Tumours: Pathology and Genetics of
Head and Neck Tumours. IARC Press: Lyon, France,
2005, p 19.
2 Chambers KJ, Lehmann AE, Remenschneider A, et al.
Incidence and survival patterns of sinonasal undiffer-
entiated carcinoma in the United States. J Neurol Surg
B 2015;76:94–100.
3 Musy PY, Reibel JF, Levine PA. Sinonasal undiffer-
entiated carcinoma: the search for a better outcome.
Laryngoscope 2002;112:1450–1455.
4 Enepekides DJ. Sinonasal undifferentiated carcinoma:
an update. Curr Opin Otolaryngol Head Neck Surg
2005;13:222–225.
5 Frierson HF Jr, Mills SE, Fechner RE, et al. Sinonasal
undifferentiated carcinoma. An aggressive neoplasm
derived from schneiderian epithelium and distinct
from olfactory neuroblastoma. Am J Surg Pathol
1986;10:771–779.
6 French CA, Kutok JL, Faquin WC, et al. Midline
carcinoma of children and young adults with NUT
rearrangement. J Clin Oncol 2004;22:4135–4139.
7 Stelow EB, Bellizzi AM, Taneja K, et al. NUT
rearrangement in undifferentiated carcinomas of the
upper aerodigestive tract. Am J Surg Pathol 2008;32:
828–834.
8 Leung SY, Yuen ST, Chung LP, et al. Epstein-Barr
virus is present in a wide histological spectrum of
sinonasal carcinomas. Am J Surg Pathol 1995;19:
994–1001.
9 Jeng YM, Sung MT, Fang CL, et al. Sinonasal
undifferentiated carcinoma and nasopharyngeal-type
undifferentiated carcinoma: two clinically, biologi-
cally, and histopathologically distinct entities. Am J
Surg Pathol 2002;26:371–376.
Figure 5 Continued.
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
VY Jo et al 9

10 Bishop JA, Guo TW, Smith DF, et al. Human
papillomavirus-related carcinomas of the sinonasal
tract. Am J Surg Pathol 2013;37:185–192.
11 Agaimy A, Koch M, Lell M, et al. SMARCB1(INI1)-
deficient sinonasal basaloid carcinoma: a novel mem-
ber of the expanding family of SMARCB1-deficient
neoplasms. Am J Surg Pathol 2014;38:1274–1281.
12 Bishop JA, Antonescu CR, Westra WH. SMARCB1
(INI-1)-deficient carcinomas of the sinonasal tract. Am J
Surg Pathol 2014;38:1282–1289.
13 Tilson MP, Bishop JA. Utility of p40 in the differential
diagnosis of small round blue cell tumors of the
sinonasal tract. Head Neck Pathol 2014;8:141–145.
14 Singh L, Ranjan R, Arava S, et al. Role of p40 and
cytokeratin 5/6 in the differential diagnosis of sinonasal
undifferentiated carcinoma. Ann Diagn Pathol 2014;18:
261–265.
15 Cerilli LA, Holst VA, Brandwein MS, et al. Sinonasal
undifferentiated carcinoma: immunohistochemical
profile and lack of EBV association. Am J Surg Pathol
2001;25:156–163.
16 Gelbard A, Hale KS, Takahashi Y, et al. Molecular
profiling of sinonasal undifferentiated carcinoma. Head
Neck 2014;36:15–21.
17 McKenna A, Hanna M, Banks E, et al. The Genome
Analysis Toolkit: a MapReduce framework for analyz-
ing next-generation DNA sequencing data. Genome Res
2010;20:1297–1303.
18 Cibulskis K, Lawrence MS, Carter SL, et al. Sensitive
detection of somatic point mutations in impure and
heterogeneous cancer samples. Nat Biotechnol 2013;
31:213–219.
19 Abo RP, Ducar M, Garcia EP, et al. BreaKmer: detection
of structural variation in targeted massively parallel
sequencing data using kmers. Nucleic Acids Res
2015;43:e19.
20 Sholl LM, Do K, Shivdasani P, et al. Institutional
implementation of clinical tumor profiling on an
unselected cancer population. JCI Insight 2016;1:
e87062.
21 Cerami E, Gao J, Dogrusoz U, et al. The cBio cancer
genomics portal: an open platform for exploring multi-
dimensional cancer genomics data. Cancer Discov
2012;2:401–404.
22 Weng AP, Ferrando AA, Lee W, et al. Activating
mutations of NOTCH1 in human T cell acute lympho-
blastic leukemia. Science 2004;306:269–271.
23 Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic
IDH1 and IDH2 mutations result in a hypermethylation
phenotype, disrupt TET2 function, and impair hema-
topoietic differentiation. Cancer Cell 2010;18:553–567.
24 Xu W, Yang H, Liu Y, et al. Oncometabolite 2-
hydroxyglutarate is a competitive inhibitor of alpha-
ketoglutarate-dependent dioxygenases. Cancer Cell
2011;19:17–30.
25 Dang L, White DW, Gross S, et al. Cancer-associated
IDH1 mutations produce 2-hydroxyglutarate. Nature
2009;462:739–744.
26 Ward PS, Patel J, Wise DR, et al. The common feature
of leukemia-associated IDH1 and IDH2 mutations is
a neomorphic enzyme activity converting alpha-
ketoglutarate to 2-hydroxyglutarate. Cancer Cell
2010;17:225–234.
27 Amary MF, Bacsi K, Maggiani F, et al. IDH1 and IDH2
mutations are frequent events in central chondrosar-
coma and central and periosteal chondromas but not in
other mesenchymal tumours. J Pathol 2011;224:
334–343.
28 Wang F, Travins J, DeLaBarre B, et al. Targeted
inhibition of mutant IDH2 in leukemia cells induces
cellular differentiation. Science 2013;340:622–626.
29 Rohle D, Popovici-Muller J, Palaskas N, et al. An
inhibitor of mutant IDH1 delays growth and promotes
differentiation of glioma cells. Science 2013;340:
626–630.
30 Liu X, Kato Y, Kaneko MK, et al. Isocitrate dehydro-
genase 2 mutation is a frequent event in osteosarcoma
detected by a multi-specific monoclonal antibody
MsMab-1. Cancer Med 2013;2:803–814.
31 Haack H, Johnson LA, Fry CJ, et al. Diagnosis of NUT
midline carcinoma using a NUT-specific monoclonal
antibody. Am J Surg Pathol 2009;33:984–991.
32 Wasserman JK, Dickson BC, Perez-Ordonez B, et al.
INI1 (SMARCB1)-deficient sinonasal carcinoma:
a clinicopathologic report of 2 cases. Head Neck Pathol
2017; e-pub ahead of print.
33 Wang K, Zhang Q, Li D, et al. PEST domain mutations
in Notch receptors comprise an oncogenic driver
segment in triple-negative breast cancer sensitive to a
gamma-secretase inhibitor. Clin Cancer Res 2015;21:
1487–1496.
34 Stoeck A, Lejnine S, Truong A, et al. Discovery of
biomarkers predictive of GSI response in triple-
negative breast cancer and adenoid cystic carcinoma.
Cancer Discov 2014;4:1154–1167.
35 Agaimy A, Daum O, Markl B, et al. SWI/SNF complex-
deficient undifferentiated/rhabdoid carcinomas of the
gastrointestinal tract: a series of 13 cases highlighting
mutually exclusive loss of SMARCA4 and SMARCA2
and frequent co-inactivation of SMARCB1 and
SMARCA2. Am J Surg Pathol 2016;40:544–553.
36 Karnezis AN, Wang Y, Ramos P, et al. Dual loss of the
SWI/SNF complex ATPases SMARCA4/BRG1 and
SMARCA2/BRM is highly sensitive and specific for
small cell carcinoma of the ovary, hypercalcaemic type.
J Pathol 2016;238:389–400.
37 Le Loarer F, Watson S, Pierron G, et al. SMARCA4
inactivation defines a group of undifferentiated thor-
acic malignancies transcriptionally related to BAF-
deficient sarcomas. Nat Genet 2015;47:1200–1205.
Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/
modpathol)
Modern Pathology (2017) 00, 1 –10
IDH2 R172X in sinonasal undifferentiated carcinoma
10 VY Jo et al