Ann Surg Oncol

https://doi.org/10.1245/s10434-020-08210-5

ORIGINAL ARTICLE – PERITONEAL SURFACE MALIGNANCY

Prognostic Molecular Classification of Appendiceal Mucinous

Neoplasms Treated with Cytoreductive Surgery
and Hyperthermic Intraperitoneal Chemotherapy
Jing Su, PhD1, Guangxu Jin, PhD2, Konstantinos I. Votanopoulos, MD, PhD3, Lou Craddock2,
Perry Shen, MD3, Jeff W. Chou, PhD1, Shadi Qasem, MD4, Stacey S. O’Neill, MD, PhD4,
Kathleen Cummins Perry, MS3, Lance D. Miller, PhD2,5, and Edward A. Levine, MD3
1
Department of Biostatistics and Data Science, Wake Forest School of Medicine, Winston-Salem, NC; 2Wake Forest
Baptist Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC; 3Surgical Oncology
Service, Department of General Surgery, Wake Forest School of Medicine, Winston-Salem, NC; 4Department of
Pathology, Wake Forest School of Medicine, Winston-Salem, NC; 5Department of Cancer Biology, Wake Forest School of
Medicine, Winston-Salem, NC

ABSTRACT assignments were evaluated by topology learning, reverse-

Background. Appendiceal mucinous neoplasm (AMN)
with peritoneal metastasis is a rare but deadly disease with
few prognostic or therapy-predictive biomarkers to guide
treatment decisions. Here, we investigated the prognostic
and biological attributes of gene expression-based AMN
molecular subtypes.
Methods. AMN specimens (n = 138) derived from a
population-based subseries of patients treated at our insti-
tution with cytoreductive surgery and hyperthermic
intraperitoneal chemotherapy (CRS/HIPEC) between
05/2000 and 05/2013 were analyzed for gene expression
using a custom-designed NanoString 148-gene panel.
Signed non-negative matrix factorization (sNMF) was used
to define a gene signature capable of delineating robustly-
classified AMN molecular subtypes. The sNMF class
Electronic supplementary material The online version of this
article (https://doi.org/10.1245/s10434-020-08210-5) contains
supplementary material, which is available to authorized users.
graph embedding and cross-cohort performance analysis.
Results. Three molecular subtypes of AMN were dis-
cerned by the expression patterns of 17 genes with roles in
cancer progression or anti-tumor immunity. Tumor subtype
assignments were confirmed by topology learning. AMN
subtypes were termed immune-enriched (IE), oncogene-
enriched (OE) and mixed (M) as evidenced by their gene
expression patterns, and exhibited significantly different
post-treatment survival outcomes. Genes with specialized
immune functions, including markers of T-cells, natural
killer cells, B-cells, and cytolytic activity showed increased
expression in the low-risk IE subtype, while genes impli-
cated in the promotion of cancer growth and progression
were more highly expressed in the high-risk OE subtype. In
multivariate analysis, the subtypes demonstrated indepen-
dent prediction power for post-treatment survival.
Conclusions. Our findings suggest a greater role for the
immune system in AMN than previously recognized. AMN
subtypes may have clinical utility for predicting CRS/
HIPEC treatment outcomes.

While rare, appendiceal cancer is associated with con-

Ó Society of Surgical Oncology 2020
siderable mortality due to the late stage at diagnosis, and
First Received: 17 September 2018 the remote likelihood of being found by screening
colonoscopic examinations.1 Although the annual inci-
L. D. Miller, PhD dence of AMN in the United States is increasing,2 it is
e-mail: ldmiller@wakehealth.edu
estimated to range from only 4503 up to 50002 cases, which
E. A. Levine, MD limits population studies. AMN all too frequently presents
e-mail: elevine@wakehealth.edu

with peritoneal dissemination. Cytoreductive surgery
(CRS) represents the primary therapeutic modality and,
when found with peritoneal surface disease, is frequently
combined with hyperthermic intraperitoneal chemotherapy
(HIPEC).4,5 Despite uniform treatment with CRS/HIPEC,
patients demonstrate widely disparate prognoses, even after
adjustment for known clinical and demographical fac-
tors.6-11 This suggests that, as in other cancers, AMN
heterogeneity at the molecular level may explain differ-
ences in tumor aggressiveness and response to CRS/HIPEC
treatment.
There is a paucity of data on the molecular genetics of
AMN. While this clearly relates to the rarity of AMN
specimens for study, another contributing factor is the low
cellularity and high mucin content characteristic of AMN,
which presents technical challenges associated with
obtaining samples with high cell purity and adequate
nucleic acid integrity. By optimizing protocols for the
latter, we recently enabled expression profiling analysis of
appendiceal adenocarcinoma using oligonucleotide
microarray technologies.9,10 In that work, we identified a
number of statistically significant prognostic gene-survival
associations and discovered two potential molecular sub-
types of AMN that could be discriminated by a 139-gene
cassette. Moreover, the subtypes exhibited significantly
different survival rates independent of known clinical and
demographical risk factors. In the current study, we
leveraged the NanoString nCounterÒ system, a highly
sensitive diagnostics platform for accurate RNA quantita-
tion, to confirm the existence of prognostic molecular
subtypes in an independent population-based cohort of 138
AMN cases treated by CRS/HIPEC at the Wake Forest
Baptist Comprehensive Cancer Center. Thus, we extend
our previous findings toward the establishment of a
molecular prognostic assay to support treatment decisions
for AMN patients.
MATERIALS AND METHODS
Patients and Tumor Specimens
All tissues and clinical data utilized in this study were
obtained for research use by patient consent under an
institutional review board-approved protocol at Wake
Forest Baptist Medical Center, Winston Salem, NC.
Selection of AMN cases was facilitated by a prospectively
maintained database of clinical and demographic infor-
mation on [ 600 appendiceal cancer patients treated with
CRS/HIPEC at Wake Forest Baptist Medical Center
(WFBMC) from 1992 to present. The technique for CRS/
HIPEC was performed in standard fashion that has been
described in detail elsewhere.9,10,12 All AMN tumor tissue
J. Su et al.
specimens acquired at the time of surgery were grossly
identified under the direction of a pathologist, processed as
formalin-fixed paraffin-embedded (FFPE) tumor blocks
and as snap frozen specimens, and maintained in a tumor
tissue repository housed within our Cancer Center’s Tumor
Tissue and Pathology Shared Resource. In the current
study, we identified a population-based sub-series of 246
CRS/HIPEC-treated patients dating from the 13-year per-
iod May 2000 to May 2013 and annotated for disease
progression or death with [ 0.5 year follow-up. Tissue
processing and analysis were conducted by our Tumor
Tissue and Pathology Shared Resource as described pre-
viously.10 Cellular content of tumor specimens was verified
by a surgical pathologist. Localized regions of tumor-
positive specimens with greatest cancer cellularity were
identified and marked for punch biopsy. A minimum
threshold of 10% cancer cellularity was observed for
qualifying samples. The same procedure was followed for
both low- and high-grade tumors. Not all available tumor
specimens yielded adequate cellularity, and 51 cases were
excluded on this basis.
AMN specimens were annotated for histological grade
(high grade versus low grade), following the Bradley sys-
tem.11 Tumors with any of the following histological
characteristics were classified as high grade: high-grade
nuclear atypia to include prominent nucleoli, hyperchro-
matic nuclei, markedly irregular nuclear membrane or
dense coarse chromatin; architectural complexity, such as
cribriform pattern, papillary formation, or extensive
nuclear stratification; high cellularity; frequent mitosis; and
signet ring cells.
NanoString n-Counter Profiling
Frozen or FFPE-processed tumor samples corresponding
to 195 AMN cases were homogenized using a Qiagen
QiaShredder and total RNA was purified using Qiagen
RNeasy Mini and RNeasy FFPE kits in our Cancer
Genomics Shared Resource. Total RNA quality require-
ments for expression profiling on the NanoString platform
included a minimum yield of 150 ng total RNA (quantified
using an Eppendorf BioPhotometer) with C 50% of the
total mass corresponding to a length of 300 base pairs or
greater as measured on an Agilent 2100 Bioanalyzer. Of
note, traditional RIN value assessment is typically not
applicable to FFPE-preserved tissues as a consequence of
prevalent RNA degradation; thus it is not considered ideal
for RNA quality control in NanoString n-Counter work-
flows. Among tumor samples with qualifying cancer
cellularity (n = 195), 37 cases were excluded from further
analysis owing to suboptimal RNA quality. Thus, 158
AMN specimens were carried forward for analysis on the
n-Counter platform. The custom n-Counter code sets
Molecular Subtyping of Appendiceal Carcinoma
   þ
Aþ W
targeted 143 genes that, by microarray analysis, showed the negative  H is solved, with H represent-
A W
most significant associations with patient overall and/or
ing the membership of samples to corresponding subtypes,
progression free survival (p \ 0.001, Cox regression
and W þ and W  representing the membership of upregu-
analysis) in our previously published studies.9 The code
lated and downregulated genes in these subtypes,
sets also targeted 5 housekeeping genes, 6 positive controls
respectively. The importance of each gene is further eval-
and 8 negative controls. The code sets were designed by
uated by its overall contribution to all subtypes using a
NanoString to target each gene’s corresponding Affymetrix
summarized membership matrix W 0 in which
probe set ‘‘target sequence’’ for consistency with the dis-
Wij0 ¼ Wijþ þ Wij . After summarization, genes that are
covery process described in our previous reports.9 RNA
significantly upregulated in a subtype and significantly
samples were profiled according to standard NanoString
downregulated in another subtype are more likely to have a
protocols on a NanoString n-Counter Analysis System
higher weight in W 0 than those that contribute to only one
operated by the Preclinical Genomic Pathology Laboratory
subtype, and thus are considered preferential genes. Before
at the University of North Carolina at Chapel Hill Line-
subtyping, genes were first screened according to their
berger Comprehensive Cancer Center. The resulting data
relevance in treatment outcomes. The significance of each
were analyzed by our Cancer Genomics Shared Resource
gene for predicting survival endpoints after CRS/HIPEC
using the NanoString n-Solver software and normalized
treatment was examined using univariate Cox proportional
according to the geometric mean of the 5 housekeeping
hazards regression. False discovery rates (FDRs) 14 were
genes (ACTB, EEF1A1, GAPDH, RPL37A, and RPLP1).
calculated to adjust for multiple testing. Among the 143
After normalization, samples with a content normalization
endogenous genes of interest, 22 were significantly asso-
factor [ 6.0 (n = 20) were deemed potentially unreliable
ciated with survival (FDR \ 0.05, Supplementary Fig. 1)
owing to poor NanoString detection, and excluded from
and thus were used as initial candidate genes for patient
further analysis. In total, tumor expression profiles from
subtyping. Tumor samples were then subjected to unsu-
138 patients (56% of the initial AMN population) were
pervised bi-clustering using sNMF and the expression data
carried forward for further analysis.
from the 22 genes. The sNMF algorithm identified 17
genes with high weight in W 0 and three AMN subtypes that
Cohort Characteristics
were well differentiated by the gene expression profiles.
Details are listed in Supplementary Table 2.
Clinical and demographic characteristics of the 138
cases are described in Table 1. Most characteristics were
Topology Analysis
proportionately similar between the low-grade and high-
grade AMN cases. However, as expected, the prognosis
The topology of patient sample heterogeneity was fur-
after CRS/HIPEC treatment differed markedly between
ther explored using manifold learning and reverse graph
low and high grade. High-grade cases exhibited shorter
embedding.15-17 The transcriptomic data of all cancer-re-
intervals to death and disease progression. Preoperative
lated or housekeeping genes over 138 patients were used
chemotherapy was administered to 64 patients (46%) of the
for principal graph learning. A latent-graph-preserved
cohort; for one patient, the use of preoperative
dimension reduction was performed using DDRTree15 and
chemotherapy could not be confirmed.
the topological heterogeneity among samples was con-
structed as a principal graph underlying the transcriptomic
Tumor Subtyping
data. Samples were clustered according to the branches of
the principal graph they were assigned to. Monocle 217 was
Transcriptional heterogeneity among patients was
used for AMN sample topology analysis.
explored using signed non-negative matrix factorization
(sNMF)—an algorithm we previously developed 13 to more
Survival Analysis
reliably discern subpopulations defined by differential gene
expression. Briefly, the data are input as a z-normalized
Both overall survival and progression-free survival were
gene expression matrix A with genes as rows and samples
analyzed for clinicopathological risk factors as well as the
as columns. Two non-negative matrices, Aþ and A , are
discovered molecular subtypes. Kaplan–Meier estimator
derived from the original input as:
  and log-rank tests were used for non-parametric survival
Aij ; ifAij [ 0 Aij ; ifAij \0 analysis. Univariate and multivariate Cox proportional
Aþij ¼ and A 
ij ¼
0; otherwise 0; otherwise hazard models were further used to evaluate the statistical
where row i indicates the i’th gene and column j indicates and clinical significance of candidate risk factors, including
the j’th sample. Instead of approximating A  WH, the non- the molecular subtypes, cancer grade, residual tumor score
 J. Su et al.

TABLE 1 Patient Total (IM = 138) Low grade (N = 76) High grade (N = 38)
characteristics
Demographics
Age–year (onset*), Mean (SD) 52.0 (12.1) 53.1(13.2) 50.9 (9.0)
Male sex-no. (%) 53 (43.4%) 30 (42.9%) 15 (44.1%)
Female sex-no. (%) 69 (56.6%) 40 (57.1%) 19 (55.9%)
Race
African-no. (%) 15 (10.9%) 13 (17.1%) 1 (2.63%)
Caucasion-no. (%) 107 (77.5%) 54 (71.1%) 34 (89.5%)
Asian-no. (%) 2 (1.45%) 2 (2.63%) 0
Others-no. (%) 2 (1.45%) 2 (2.63%) 0
Ethnicity
Hispanic or Lartino-no. {%) 3 (2.17%) 3 (3.95%) 0
Others-no. (%) 136 (97.83%) 73 (96.05%) 48 (100%)
Overall Survival: no. 135 76 38
Events-no. (%) 65 (48.1%) 23 (30.3%) 32 (84.2%)
Followup (yr), Mean (SD) 2.95 (2.14) 3.87(2.21) 1.48 (1.01)
Time of event (yr), Mean (SD) 1.96 (1.68) 2.91 (2.21) 1.43 (1.00)
Progression-free survival: no. 80 53 19
Events-no. (%) 54 (67.5%) 33 (62.3%) 18 (94.7%)
Followup (yr), Mean (SD) 2.51 (2.16) 2.96(2.30) 1.03 (0.713)
Time of event (yr), mean (SD) 1.36(0.997) 1.63(1.13) 0.907 (0.497)
Surgical score R 126 71 34
R0/R1-no. (%) 50 (39.7%) 26 (36.6%) 11 (32.3%)
R2-no. (%) 76 (60.3%) 45 (63.4%) 23 (67.6%)
ECOG performance status 122 70 33
Grade 0-no. (%) 64 (52.5%) 33 (47.1%) 16 (48.5%)
Grade 1-no. (%) 46 (37.7%) 31 (44.3%) 11 (33.3%)
Grade 2-no. (%) 10 (8.20%) 5 (7.14%) 5 (15.2%)
Grade 3-no. (%) 2 (1.64%) 1 (1.43%) 1 (3.03%)
Preoperative chemotherapy-no. (%) 64 (46.4%) 21 (27.6%) 34 (89.5%)
Adjuvant chemotherapy-no. (%) 39 (28.2%) 18 (23.7%) 16 (42.1%)

(R), ECOG score (Eastern Cooperative Oncology Group

TABLE 2 Multivariate cox regression for overall survival analysis
Scale of Performance Status), and patients’ age and sex.
Hazard 95% Confidence P
ratio interval
Robustness Analysis of the 17-Gene Signature
Grade 6.6 2.8, 15 1.2e-05
ECOG score 1.4 0.89, 2.3 0.14 We examined the robustness of the 17-gene signature
R score 2.7 1.3, 5.7 0.009 and the prognostic attributes of the AMN subtypes in an
Adjuvant chemo 0.68 0.34, 1.3 0.27 independent patient population of low- and high-grade
Preoperative chemo 1.4 0.63, 3.0 0.42 AMN (n = 40) profiled on the Affymetrix U133A Gene-
Age 0.99 0.97, 1.0 0.64 Chip system, which was previously published by our
Sex (M) 0.89 0.48, 1.7 0.72 team.9 Briefly, the expression of the 17 signature genes
OE subtype 2.5 1.0, 6.2 0.044 discovered in this work were scaled and used to objectively
M subtype 1.9 0.77, 4.6 0.177 classify patients into the corresponding AMN subtypes
using the sNMF approach with the same parameter set-
n = 98, number of events = 46 Likelihood ratio test: p = 2e-9
tings. The overall survival rates of patients partitioned into
Bold texts indicate statistically significant risk factors with p-values
the AMN subtypes were compared to examine the prog-
less than 0.05
nostic power of the 17-gene signature.
Molecular Subtyping of Appendiceal Carcinoma

RESULTS (EPCAM, SPINK1, ESRP1, CLDN3, CLDN4, ELF3,

AMN tumor gene expression profiles were generated on
the NanoString gene panel and analyzed by the sNMF
algorithm. A 17-gene signature, composed of 7 immune
genes and 10 cancer-associated genes, was identified that
could robustly classify the 138 patients into three distinct
molecular subtypes, termed immune-enriched (IE, n = 40),
oncogene-enriched (OE, n = 43), and mixed (M, n = 55)
(Fig. 1). These subtypes showed significantly different
gene expression patterns and post-treatment survival out-
comes. Genes with specialized functions in lymphocyte
physiology, including markers of T-cells (TRA, TRBC1)
natural killer cells (KLRF1, KLRG1) and cytolytic activity
(PRF1), exhibited elevated expression levels in association
with the more favorable outcomes of IE, consistent with
heightened effector cell infiltration and protective anti-tu-
mor function in these tumors. By contrast, genes implicated
in the promotion of cancer growth and progression
GPX2) were more highly expressed in the poor outcome-
associated OE group, consistent with a more aggressive
tumor growth phenotype.
The inter-tumor heterogeneity in terms of gene expres-
sion patterns among AMN samples were further explored
using reversed graph embedding. As demonstrated in
Fig. 2, in a typical gene expression topology analysis,
tumor heterogeneity was visualized from three perspec-
tives: (1) local similarity: where transcriptionally similar
samples were visualized as data points positioned close to
each other; (2) global heterogeneity: where gradual chan-
ges in gene expression patterns across the whole cohort
were visualized as trend lines (black lines) across data
points (samples); and (3) hierarchical clusters: where
major clusters were represented as data points around long
branches, with minor clusters around short branches.

Patient Subtypes
Genes Immune Enriched Oncogene Enriched Mixed
IL23A
Immune Genes

CD37
KLRF1
MAFB
PRF1
TRA
TRBC1
CLDN3
CLDN4
ELF3
Oncogenes

EPCAM
GPX2
KRT20
LGALS4
PHGR1
ESRP1
SPINK1

Gene Expression
–4 –2 0 2 4

Clinical Features
Grade
ECOG Score
R Score

Annotation
Grade: Low High ECOG Score: E0 E1 E2 E3 R Score: R0 R0/R1 R1 R2a R2b R2c

FIG. 1 AMN molecular subtypes defined by sNMF. Three identified
AMN molecular subtypes (immune enriched, oncogene enriched, and
mixed) and 17 associated signature genes (7 immune genes and 10
oncogenes) displayed with clinical features (grade, ECOG score, and
R score) are shown. Top: heatmap of the normalized gene expression
patterns (rows) corresponding to tumor samples comprising the three
molecular subtypes (columns). Bottom: patient clinical features
(grade, ECOG score, and R score) corresponding to tumor samples
in heatmap columns are shown
 J. Su et al.

(A) Transcriptomic Landscape of AMN Patients

Topological Classes Subtypes by sNMF

Class: OE IE M Subtype: OE IE M

(B) Expression of Feature Genes

EPCAM PRF1
2
1

Normalized Expression Level

Normalized Expression Level

1
0

0
–1

–1
–2

–2

0 2 4 6 8 0 2 4 6 8
Pseudo-location on Trajectory Pseudo-location on Trajectory

FIG. 2 Gene expression topology graphs of AMN patients.
Transcriptomic similarities among AMN samples are shown
represented by circles (AMN samples), distance (similarity of
samples) and trend line trajectories (global heterogeneity among
samples). Trend line segments represent distinct hierarchical clusters.
a Patient samples (circles) are colored according to topology class
assignments (left panel) or sNMF molecular subtype class
assignments (right panel). b The changes of relative expression
levels of selected genes (y-axis) along the trajectory, from OE toward
IE (x-axis). EPCAM (OE gene member), left panel. PRF1 (IE gene
member), right panel

This analysis (Fig. 2a, left) uncovered three dominating
topological classes (represented by data points around the
three long black lines) and two minor classes (the two short
black lines). Among them, two (data points colored in
orange and purple) represented the high-risk OE subtypes,
one (gray data points) for the intermediate-risk M subtype,
and two (green and blue data points) for the low-risk IE
subtype. To examine whether the discovered molecular
subtypes were consistent with the sNMF-based subtyping
results, we labeled the sNMF-based subtyping results onto
the learned gene expression topology graph (Fig. 2a, right).
The results of the two subtyping approaches were highly
consistent (Fig. 2a). The Jaccard similarities between the
IE, OE, and M subtypes and the corresponding topological
clusters were 0.85, 0.84, and 0.73, respectively.
Molecular Subtyping of Appendiceal Carcinoma

We further examined whether the gene expression pat- 40-patient AMN cohort 9 profiled on the Affymetrix Gen-
terns in the three molecular subtypes discovered by the eChip platform. Three AMN subtypes were again
sNMF approach were consistent with the gene expression discerned by the signature, and the differential survival
topology analysis. We used EPCAM and PRF1 as exam- rates of the resulting AMN subtypes (p B 2e-05) closely
ples. In Fig. 2b, points represent the normalized gene mirrored those observed for the 138-patient cohort, thus
expression levels in the corresponding samples along the demonstrating a robust performance of the signature across
trajectory shown in Fig. 2a, starting from the OE end patient populations and RNA quantitation platforms.
toward the IE end. Moving along the branches from the The molecular subtypes demonstrated independent pre-
high-risk OE cluster (left branch) toward the low-risk IE diction power for post-treatment survival (Table 2). Using
cluster, the expression of oncogenes (e.g., EPCAM) multivariate Cox regression, we adjusted the prediction
declined, while the immune activities (e.g., PRF1) power of the subtypes by removing effects of other clinical
increased. and demographical factors including cancer grade (low and
These findings from the transcriptomic topology analy- high), performance status (ECOG score), residual tumor
sis indicated that the discovered subtypes were score (R score, grouped as R0/R1 and R2), preoperative
reproducible by different subtyping methods, that they chemotherapy, adjuvant chemotherapy and patients’ age
represented the majority of the heterogeneity of the AMN and sex. The molecular subtypes remained a significant risk
population, and that the discovered gene expression pat- factor in the model (p B 0.044, hazard ratio: 2.5) together
terns were consistent across the topological graph. with cancer grades (p B 1.2e-05, hazard ratio: 6.6) and R
The overall survival and progression-free survival of the scores (p B 0.009, hazard ratio: 2.7).
discovered subtypes (Fig. 3) differed significantly (log-
rank tests with p B 1.3e-06 and p B 2.0e-04, respectively). DISCUSSION
Three-year overall survival rates were 83% (95% CI
71.5%–96.5%), 55% (43%–72%), and 36% (24%–55%), Accurate patient prognosis plays an essential role in
and 3-year progression survival rates were 65.5% (51%– precision oncology.18,19 In AMN, the predominant prog-
84%), 30% (17%–53%), and 14% (5.0%–41%) for IE, M, nostic variables associated with CRS/HIPEC outcomes are
and OE subtypes, respectively. To test the robustness of the histologic grade, completeness of cytoreduction (R score)
17-gene signature for delineating prognostic AMN sub- and patient performance status (ECOG score). In this study,
types, we objectively applied the sNMF algorithm we confirm that molecular subtypes of AMN, defined by
comprising the 17 genes to our previously reported gene signatures reflective of effector immune cell

K–M Estimate of Overall Survival K–M Estimate of Progression-free Survival K–M Estimate of Overall Survival
1.0

1.0

1.0

Subtype Subtype Subtype

IE (Low Risk) IE (Low Risk) IE (Low Risk)
OE (High Risk) OE (High Risk) OE (High Risk)
0.8

M (Inter Risk)
0.8

M (Inter Risk)
0.8

M (Inter Risk)
Survival Function

Survival Function

Survival Function
0.6

0.6

0.6
0.4

0.4

0.4
0.2

0.2

0.2
0.0

0.0

0.0

Log-rank p-value: 1.34 e-06 Log-rank p-value: 2.02 e-04 Log-rank p-value: 2e–05

0 2 4 6 8 10 0 2 4 6 8 10 0 2 4 6 8
Time (yr) Time (yr) Time (yr)
Subtype n events Median (yr, 95%Cl) Subtype n events Median (yr, 95%Cl) Subtype n events Median (yr, 95%Cl)
OE (High Risk) 41 29 1.4 (1.1,3.4) OE (High Risk) 21 19 0.9 (0.6,2.0) OE (High Risk) 11 11 0.6 (0.5, –)
IE (Low Risk) 40 8 – IE (Low Risk) 32 15 4.1 (3.1, –) IE (Low Risk) 13 3 –
M (Inter Risk) 54 28 3.5 (2.4, –) M (Inter Risk) 27 20 1.5 (1.1,3.5) M (Inter Risk) 16 11 1.9 (1.1, –)
Log-rank p-value: 1.3 e-06 Log-rank p-value: 2.0 e-04 Log-rank p-value: 2 e-05

FIG. 3 Analysis of survival differences between AMN subtypes. The estimator. Statistical metrics about these survival profiles including
overall (left) and progression-free (middle) survival curves of the sample size, number of events, and median survival time with 95%
three identified subtypes (IE: immune enriched, OE: oncogene confidence interval are summarized in the corresponding
enriched, M: mixed) in the reported cohort as well as the overall Tables beneath survival plots. Log-rank tests were used to evaluate
survival curves (right) of a 40-patient AMN cohort profiled on the the survival differences between AMN subtypes
Affymetrix GeneChip platform were estimated by Kaplan–Meier

infiltration and tumor oncogenic properties, carry signifi-
cant additive prognostic value not provided by
conventional prognostic markers. In our previous expres-
sion profiling study 9 which utilized Affymetrix
oligonucleotide arrays, unbiased hierarchical clustering
analysis initially delineated three AMN molecular subtypes
based largely on the expression characteristics of the
oncogene-enriched genes. The highly expressed subtype
was associated with poor survival outcomes, while the
subtypes with intermediate and low expression were
associated with better survival outcomes. Further clustering
of the tumors based on a 139-gene cassette yielded only
two predominant subtypes that exhibited significant sur-
vival differences, with the intermediate tumors dispersed
among the two subtypes. The re-assignment of the inter-
mediate-expressing subtype to the two predominant
subtypes owed to the nature of the clustering algorithm and
the relatively small sample size investigated in that study.
In the current study, however, the sNMF algorithm also
identified a distinguishable intermediate-expressing sub-
type [i.e., equating with the mixed (M) subtype] with a
survival rate intermediate to that of the OE and IE sub-
types, confirming in this larger study the existence of a
clinically relevant mixed expression subtype. Consistent
with our previous findings, multivariate Cox regression
analysis showed an independent relationship between the
subtype classes and overall survival. The high-risk OE
subtype exhibited a hazard ratio that was exceeded only by
histologic grade, highlighting its clinical relevance as a
significant prognostic biomarker in this setting. Further-
more, the prognostic attributes of the AMN molecular
subtypes discerned by the 17-gene signature were found to
be highly reproducible in our previously reported 40-pa-
tient AMN cohort. This indicates a robust level of
portability of the signature across patient populations and
RNA quantitation platforms (NanoString n-Counter and
Affymetrix GeneChip microarray). Thus, our findings
confirm the reproducible and independent significance of
gene-survival associations in AMN.9,10
In this work, we identified an optimal set of 17 genes for
partitioning AMN cases into IE, OE, and M prognostic
subtypes. The genes comprising our model suggest that
AMN is cellularly heterogeneous, with a complex immune,
stromal and cancer cell composition that underlies patient
outcomes. The association between the low-risk IE subtype
and the heightened expression of genes with specialized
roles in effector immune cell function suggests a role for
immune-mediated control of AMN progression in some
patients. In these tumors, further investigation of the
functional and transcriptional dynamics of the AMN tumor
microenvironment would more fully elucidate the cellular
complexity of AMN underlying its pathogenesis, progres-
sion and tumor-immune interactions.
J. Su et al.
Cohort size is always a critical factor in studies that
investigate tumor heterogeneity, but even more so in rare
diseases such as AMN. Our study cohort (n = 138) is the
largest to date for AMN genomics profiling with clinical
follow-up and has enabled the discovery of reliable
molecular subtypes of AMN. The consistency observed
between two independent clustering approaches, as well as
the distinct prognostic survival differences observed, sug-
gests that the major AMN subtypes definable by gene
expression patterns were discovered in this analysis.
One limitation of this study was the relatively small
sample size associated with high-grade AMN (n = 38). As
demonstrated in Supplementary Fig. 2, the three molecular
subtypes exhibited distinct survival profiles among the
patients with low-grade disease (n = 76). However, in the
context of high-grade disease, the survival function of the
IE subtype (n = 6) could not be sufficiently established.
Further analysis of subtypes in a larger population of high-
grade AMN is warranted.
As in other tumor systems, the risk-related molecular
subtypes described here hold promise for application in
clinical decision support. To be implemented clinically,
prognostic gene expression assays must be both reliable
and facile. This takes into account consideration for both
the RNA assay platform and the reliability of the sample
processing parameters. The nCounterÒ Analysis System
requires as little as 25 ng of total RNA, requires less than
15 min hands-on operation, and generates prognostic out-
comes within 24 h. Our findings support the feasibility of
using the nCounterÒ platform for the prognostic molecular
subtyping of AMN patients. AMN surgical tissues are
cellularly heterogeneous, marked by frequently low or
dispersed tumor cellularity and diverse cellular and acel-
lular stromal features. How this impacts the reliability of
AMN molecular subtyping is a question of optimal sample
processing methodology that warrants further investiga-
tion. Strategies to incorporate AMN subtyping into the
clinical management of AMN is the focus of ongoing work
and will require multi-institutional validation studies.
Whether the OE, IE and M subtypes benefit from dif-
ferent therapeutic approaches is an attractive hypothesis.
Clearly, our current findings cannot address this issue;
however, the OE subtype seems a good candidate for
preoperative chemotherapy trials and the IE for preopera-
tive immunotherapy studies.
ACKNOWLEDGEMENTS The authors acknowledge the
DEMON high performance computing cluster, the Greenplum mas-
sively parallel processing database and the Data Lake cloud storage
and computing facility at Wake Forest University School of Medi-
cine, the Texas Advanced Computing Center (TACC) at The
University of Texas at Austin (http://www.tacc.utexas.edu), and the
Extreme Science and Engineering Discovery Environment (XSEDE,
which is supported by National Science Foundation grant number
Molecular Subtyping of Appendiceal Carcinoma
ACI-1548562), for providing high performance computing resources
that have contributed to the research results reported within this
paper.
FUNDING This work was supported, in part, by the Orin Smith
Family fund (to E.A.L.), pilot funds from the National Organization
for Rare Disorders (to E.A.L. and L.D.M.) and the Wake Forest
Baptist Compressive Cancer Center’s Shared Resources: Cancer
Genomics (CGSR), Tumor Tissue & Pathology (TTPSR) and
Bioinformatics (BISR) supported by the National Cancer Institute’s
Cancer Center Support Grant award number P30CA012197. The
content of this publication is solely the responsibility of the authors
and does not necessarily represent the official views of the National
Cancer Institute.
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