Mucinous and neuroendocrine breast carcinomas are transcriptionally distinct from invasive ductal carcinomas of no special type

ArticleinModern Pathology 22(11):1401-14 · July 2009with75 Reads
Impact Factor: 6.19 · DOI: 10.1038/modpathol.2009.112 · Source: PubMed
Abstract

Mucinous carcinoma is considered a distinct pathological entity. However, mucinous tumours can be divided into a least two groups: mucinous A (or paucicellular) and mucinous B (or hypercellular). Mucinous B cancers display histological features that significantly overlap with those of neuroendocrine carcinomas. We investigate using genome-wide oligonucleotide microarrays whether mucinous A, mucinous B and neuroendocrine carcinomas are entities distinct from histological grade- and molecular subtype-matched invasive ductal carcinomas of no special type. Mucinous A and B and five neuroendocrine carcinomas were of luminal A subtype, whereas one neuroendocrine tumour was of luminal B phenotype. When analysed in conjunction with grade- and molecular subtype-matched invasive ductal carcinomas, hierarchical clustering analysis showed that the majority of mucinous and neuroendocrine cancers formed a separate cluster. Significance analysis of microarrays identified 3155 genes differentially expressed between mucinous/ neuroendocrine carcinomas and grade- and molecular subtype-matched invasive ductal carcinomas (false discovery rate <0.85%), and revealed that genes associated with connective tissue/extracellular matrix were downregulated in mucinous/neuroendocrine cancers compared to invasive ductal carcinomas. When subjected to hierarchical clustering analysis separately, mucinous A cancers formed a discrete subgroup, whereas no separation was observed between mucinous B and neuroendocrine cancers. In fact, significance of microarray analysis showed no transcriptomic differences between mucinous B and neuroendocrine cancers, whereas mucinous A cancers displayed 89 up- and 26 downregulated genes when compared with mucinous B (false discovery rate <1.15%) and 368 up- and 48 downregulated genes when compared to neuroendocrine carcinomas (false discovery rate <1.0%). Our results provide circumstantial evidence to suggest that mucinous and neuroendocrine carcinomas are transcriptionally distinct from histological grade- and molecular subtype-matched invasive ductal carcinomas, and that luminal A breast cancers are a heterogeneous group of tumours. These findings support the contention that mucinous B and neuroendocrine carcinomas are part of a spectrum of lesions, whereas mucinous A is a discrete entity.

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Mucinous and neuroendocrine breast
carcinomas are transcriptionally distinct from
invasive ductal carcinomas of no special type
Britta Weigelt
1,3
, Felipe C Geyer
2
, Hugo M Horlings
1
, Bas Kreike
1
, Hans Halfwerk
1
and Jorge S Reis-Filho
2
1
Division of Experimental Therapy, The Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands
and
2
Molecular Pathology Laboratory, The Breakthrough Breast Cancer Research Centre, Institute of Cancer
Research, London, UK
Mucinous carcinoma is considered a distinct pathological entity. However, mucinous tumours can be divided
into a least two groups: mucinous A (or paucicellular) and mucinous B (or hypercellular). Mucinous B cancers
display histological features that significantly overlap with those of neuroendocrine carcinomas. We investigate
using genome-wide oligonucleotide microarrays whether mucinous A, mucinous B and neuroendocrine
carcinomas are entities distinct from histological grade- and molecular subtype-matched invasive ductal
carcinomas of no special type. Mucinous A and B and five neuroendocrine carcinomas were of luminal A
subtype, whereas one neuroendocrine tumour was of luminal B phenotype. When analysed in conjunction with
grade- and molecular subtype-matched invasive ductal carcinomas, hierarchical clustering analysis showed
that the majority of mucinous and neuroendocrine cancers formed a separate cluster. Significance analysis of
microarrays identified 3155 genes differentially expressed between mucinous/ neuroendocrine carcinomas and
grade- and molecular subtype-matched invasive ductal carcinomas (false discovery rate o0.85%), and revealed
that genes associated with connective tissue/extracellular matrix were downregulated in mucinous/
neuroendocrine cancers compared to invasive ductal carcinomas. When subjected to hierarchical clustering
analysis separately, mucinous A cancers formed a discrete subgroup, whereas no separation was observed
between mucinous B and neuroendocrine cancers. In fact, significance of microarray analysis showed no
transcriptomic differences between mucinous B and neuroendocrine cancers, whereas mucinous A cancers
displayed 89 up- and 26 downregulated genes when compared with mucinous B (false discovery rate o1.15%)
and 368 up- and 48 downregulated genes when compared to neuroendocrine carcinomas (false discovery rate
o1.0%). Our results provide circumstantial evidence to suggest that mucinous and neuroendocrine carcinomas
are transcriptionally distinct from histological grade- and molecular subtype-matched invasive ductal
carcinomas, and that luminal A breast cancers are a heterogeneous group of tumours. These findings support
the contention that mucinous B and neuroendocrine carcinomas are part of a spectrum of lesions, whereas
mucinous A is a discrete entity.
Modern Pathology (2009) 22, 1401–1414; doi:10.1038/modpathol.2009.112; published online 24 July 2009
Keywords:
breast cancer; histological type; microarrays; classification
Pure mucinous carcinoma of the breast is a rare
histological type, which accounts for approximately
2% of all invasive breast cancers and is charac-
terised by clusters of tumour cells floating in large
amounts of extracellular mucus.
1
Neuroendocrine
carcinoma represents 2–5% of invasive breast
cancers and displays morphological features similar
to those of neuroendocrine neoplasms of other
organs, including the gut.
1
Both mucinous and neuroendocrine carcinomas
are recognised as distinct histological entities in the
latest edition of the World Health Organisation
Received 13 May 2009; revised and accepted 26 June 2009;
published online 24 July 2009
Correspondence: Dr B Weigelt, PhD, Cancer Research UK, London
Research Institute, Lincoln’s Inn Fields Laboratories, Room 224,
44 Lincoln’s Inn Fields, London WC2A 3PX, UK or Dr JS
Reis-Filho, MD, PhD, FRCPath, The Breakthrough Breast Cancer
Research Centre, Institute of Cancer Research, 237 Fulham Road,
London SW3 6JB, UK.
E-mail: Britta.Weigelt@cancer.org.uk or Jorge.Reis-Filho@icr.ac.uk
3
Current address: Signal Transduction Laboratory, Cancer
Research UK, London Research Institute, London, UK.
Modern Pathology (2009) 22, 14011414
& 2009 USCAP, Inc. All rights reserved 0893-3952/09 $32.00 1401
www.modernpathology.org
Page 1
(WHO) classification of breast neoplasms.
1,2
Capella
et al,
3
however, reported that pure mucinous carci-
noma of the breast is not a single homogeneous
entity, but comprises two main subtypes based on
structural and cytological features: mucinous A (or
paucicellular), which represents the ‘classical’ var-
iant with large quantities of extracellular mucin, and
mucinous B (or hypercellular) tumours, which con-
tain less mucin and often show neuroendocrine
differentiation and argyrophilia.
3
Neuroendocrine
carcinoma has also been suggested to not constitute
a single clinicopathological entity, and several histo-
logical subtypes have been described including the
cellular mucinous type,
4–9
which could also be
classified as part of the spectrum of mucinous lesions
(ie mucinous B). In addition, some have suggested
that neuroendocrine differentiation may represent a
pathway of neoplastic development in a range of
breast cancers,
10
although its biological and clinical
behaviour remains a matter of contention.
11–14
The advent of expression profiling analysis has led
to the development of a working model for a breast
cancer molecular taxonomy comprising five mole-
cular subtypes (ie luminal A, luminal B, basal-like,
HER2 þ and normal breast-like).
15
This classification
has shown to be of prognostic significance: the
luminal A group of tumours have good outcome,
whereas the luminal B, HER2 þ and basal-like
groups have a significantly worse prognosis.
16,17
Each molecular subtype, however, may constitute a
heterogeneous group of cancers with distinct tran-
scriptomic and genomic characteristics, clinical
behaviour and drug response. This has been com-
prehensively shown for basal-like breast cancers,
18–28
which are heterogeneous at the clinical, histological
and molecular levels; however, only few studies
addressed the diversity of other molecular subtypes.
Recent studies have shown that both mucinous
and neuroendocrine tumours consistently pertain to
the luminal molecular subtype;
19,29
however, the
similarities and differences between these entities at
the molecular level has thus far not been explored.
The aims of this study were twofold: (i) to determine
whether mucinous and neuroendocrine carcinomas
are molecular entities distinct from histological
grade- and molecular subtype-matched invasive
ductal carcinomas of no special type; and (ii) to
define whether the histological entities mucinous
and neuroendocrine carcinomas are distinct at
the transcriptomic level given the overlapping
morphological features between mucinous A and
mucinous B and between mucinous B and neuroen-
docrine breast cancers.
Materials and methods
Samples
Mucinous and neuroendocrine carcinomas
Consecutive samples of tumours classified as pure
mucinous and neuroendocrine carcinomas were
selected from the frozen tissue bank of The Nether-
lands Cancer Institute/Antoni van Leeuwenhoek
hospital (NKI/AVL). Before and after cutting tissue
sections for RNA extraction, a representative section
was stained with haematoxylin and eosin and semi-
quantitatively assessed for the percentage of tumour
areas over the total sample area. Only samples
containing Z60% tumour cells were selected for
downstream analysis. The transcriptomic profiles of
the cases reported in this study were published in
part in an earlier study.
19
Gene expression data are
publicly available at ArrayExpress (E-NCMF-3).
Control group (invasive ductal carcinomas of no
special type)
We retrieved gene expression data from 102 invasive
breast carcinomas that were part of an unrelated
research project in our institute and were published
in part by Kreike et al.
18,30
This gene-expression
dataset differs only in tumour type analysis (ie
predominantly invasive ductal carcinomas), but is
similar with regards to experimental work-up. Of
these 102 invasive breast carcinomas, 91 were
invasive ductal carcinomas of no special type (hence-
forward ‘invasive ductal carcinomas’) and used as
controls. Gene expression data are publicly available
at ArrayExpress (E-NCMF-24). Detailed information
on RNA extraction, amplification, labelling, hybridi-
sation, scanning, microarray platform and analysis
has been described earlier.
18,19
Histopathological Review and Immunohistochemistry
All samples of mucinous and neuroendocrine
carcinomas were independently reviewed by two
pathologists (FCG and JSR-F). Tumours were classi-
fied as of mucinous histological type according to
the criteria outlined by Ellis et al
1
and subclassified
into mucinous A and mucinous B according to the
criteria outlined by Capella et al.
3
In brief, mucinous
A tumours were paucicellular and 60–90% of the
tumour areas were composed of extracellular mucin
in which scattered neoplastic cells arranged in
trabeculae, ribbons, festoons and rings were found.
These neoplastic cells either lacked or only rarely
showed intracellular mucin. Mucinous B tumours
had a higher cellularity, a lower mucin content
(30–75% of tumour area), and were preferentially
characterised by neoplastic cells, often containing
intracytoplasmic mucin, arranged in large, densely
packed clumps and sheet-like structures.
3
Neuroen-
docrine carcinomas were defined according to the
WHO criteria. In brief, tumours displayed morpho-
logical features reminiscent of those of neuroendo-
crine carcinomas of gastrointestinal tract and lungs,
including tumour cells arranged in solid nests and/
or trabeculae separated by delicate fibrovascular
stroma. Rosettes, peripheral palisading and solid
papillary formations were also considered features
of neuroendocrine cancers. To establish an objective
Mucinous and neuroendocrine carcinomas
1402 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 2
diagnosis, neuroendocrine carcinomas were diag-
nosed only when usual morphological features and
expression of at least one neuroendocrine marker (eg
chromogranin, synaptophysin or CD56) in at least
50% of cells were found.
1
A perfect agreement
between the two pathologists was reached in all but
in one case (3683). The discrepancy was resolved by
simultaneous analysis of representative histological
sections of this tumour on a multi-headed micro-
scope. The pathological characteristics of all cases
are presented in Table 1.
Immunohistochemical analysis of mucinous and
neuroendocrine carcinomas was performed as de-
scribed earlier
19
with antibodies raised against
chromogranin (DAK-A3, 1:8000, Dako), synaptophy-
sin (polyclonal, 1:400, Dako) and CD56 (123C3,
31
1:800). For chromogranin and synaptophysin only
cytoplasmic staining was considered as specific, for
CD56 both membrane and cytoplasmic expression.
Out of the 91 invasive ductal carcinomas, none
displayed overt features of mucinous or neuroendo-
crine differentiation in 410% of the tumour areas.
Owing to the lack of material available from the
invasive ductal carcinomas, immunohistochemical
analysis of the neuroendocrine markers chromogra-
nin, synaptophysin and CD56 was not performed on
these control tumours.
Data Analysis
A subset of the 34 580 probes was selected, based
on the following criteria: unambiguous mapping
information for the probe, expression data available
for at least 75% of all experiments and the expres-
sion level significantly different from the reference
expression in at least 10% of experiments with a
P-value of o0.01. These criteria reduced the total
number of transcripts to 8398 significantly regulated
transcripts.
To define whether a tumour pertained to basal-
like, luminal A, luminal B, HER2 or normal breast-
like molecular subgroup, we determined the Spear-
man’s rank correlation of each case with the
‘Intrinsic/UNC’ class centroids by Hu et al.
17
Almost
all ‘intrinsic genes’ were identified (293 out of 306
unique genes).
After defining the molecular subtypes of the
invasive ductal carcinomas, a subset of 24 of these
tumours were selected to match with the mucinous
A, mucinous B and neuroendocrine carcinomas
based on histological grade and molecular subtype.
For unsupervised clustering analysis, we per-
formed average-linkage hierarchical clustering of a
centred correlation similarity matrix of the muci-
nous, neuroendocrine carcinomas and invasive
ductal carcinomas (23 pertaining to the luminal A
and 1 to the luminal B subtype) with 8398 filtered
genes using the program Cluster,
32
and results were
visualised with TreeView. Genes and arrays were
median centred.
To determine the significantly differentially ex-
pressed genes between mucinous A, mucinous B,
neuroendocrine and grade- and molecular subtype-
matched invasive ductal carcinomas, we used
significance analysis of microarrays software
33
per-
forming 1000 permutations, as described earlier.
20
Exact Hypergeometric Probability Analysis for Gene
List Enrichment
Analysis for enrichment of differentially expressed
genes identified by significance analysis of micro-
arrays was performed using hypergeometric probabil-
ity analysis, whereby the number of genes in common
between two groups was identified and a representa-
tion factor (the number of overlapping genes divided
by the expected number of overlapping genes drawn
from two independent groups) was calculated. A
representation factor 41 indicates more overlap than
expected of two independent groups, whereas a
representation factor o1 indicates less overlap than
expected. The probability of finding an overlap of
that number of genes was then calculated using
the hypergeometric probability formula: C(D, x)*
C(ND, nx)/C(N,n). (http://elegans.uky.edu/MA/
progs/representation.stats.html).
Ingenuity Pathway Analysis
To determine pathways and networks that were
significantly regulated in the gene expression data of
mucinous, neuroendocrine tumours and grade- and
molecular subtype-matched invasive ductal carcino-
mas, we performed pathway analysis using the
Ingenuity Pathway Analysis program (http://www.
ingenuity.com). The differentially expressed genes as
identified by significance analysis of microarrays were
mapped to networks available in the Ingenuity
database and were ranked by score. The score
indicates the likelihood of the genes in a network
being found together because of random chance. Using
a 99% confidence level, scores of Z3 are significant.
Results
Mucinous and Neuroendocrine Carcinomas are
Molecularly Distinct from Grade- and Molecular
Subtype-Matched Invasive Ductal Carcinomas of no
Special Type
Out of 24 cases, six were classified as neuroendo-
crine carcinomas and 18 as mucinous carcinomas
according to the WHO criteria.
1
Mucinous cancers
were subclassified into A and B subgroups accord-
ing to the criteria of Capella et al:
3
10 were bona fide
mucinous A and 8 were mucinous B; no case was
classified as mixed (Figure 1; Table 1). All neuroen-
docrine carcinomas displayed features consistent
with the diagnosis of solid neuroendocrine carcinoma
of the breast
1
and expressed at least one marker
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1403
Modern Pathology
(2009) 22, 14011414
Page 3
Table 1 Histological characteristics and molecular subtypes according to the ‘Intrinic/UNC’ centroids (Hu et al, BMC Genomics 7, 2006) of mucinous and neuroendocrine carcinomas
Case Extracellular
mucin
Nuclear
pleomorphism
Histological
grade
Structure pattern Cytological pattern Foamy
cells
Intracellular
mucin
Signet ring
cells
Apocrine
features
Synapto-
physin
Chromo-
granin
CD56 Histological
type
Molecular
subtype
3671 80% 3 2 Rings, festoons, ribbons,
trabeculae
Polyedrical Yes No No Diffuse 0 0 0 Mucinous A Luminal A
3672 80% 2 1 Ribbons, festoons, rings Polyedrical, columnar
with apical snouts
Yes Rare No Focal 0 0 0 Mucinous A Luminal A
3673 85% 2 1 Rings, ribbons,
cribriform
Columnar with apical
snouts, spindle
No No No No 1+ 0 0 Mucinous A Luminal A
3674 60% 3 1 Rings, cribriform,
festoons, sheets
Polyedrical, columnar
with apical snouts
Yes No No No 0 0 1+ Mucinous A Luminal A
3675 80% 2 1 Ribbons, rings, festoons Polyedrical, columnar
with apical snouts
No Yes Yes No 0 0 0 Mucinous A Luminal A
3676 80% 3 1 Rings, ribbons Polyedrical, hyaline Yes Yes Yes No 1+ 0 0 Mucinous A Luminal A
3677 80% 2 1 Trabeculae, rings,
festoons
Columnar with apical
snouts, spindle
Yes Yes Rare No 0 0 0 Mucinous A Luminal A
3678 70% 3 1 Cribriform, festoons Polyedrical, columnar
with apical snouts
Yes Yes Yes Diffuse 0 0 0 Mucinous A Luminal A
3679 90% 2 1 Rings, festoons, ribbons,
trabeculae
Columnar with apical
snouts, spindle
No Yes No Focal 0 0 0 Mucinous A Luminal A
3680 90% 2 1 Festoons, cribriform,
ribbons, sheets
Polyedrical No Yes Yes No 0 0 0 Mucinous A Luminal A
3681 30% 3 3 Sheets, trabeculae Polyedrical No No No No 0 0 0 Mucinous B Luminal A
3682 30% 3 1 Sheets, cribriform Spindle, hyaline No No No No 1+ 1+ 1+ Mucinous B Luminal A
3684 30% 2 1 Sheets Spindle, hyaline No No No No 0 0 0 Mucinous B Luminal A
3685 60% 3 1 Sheets, cribriform Cuboidal, spindle Yes No No No 0 0 0 Mucinous B Luminal A
3686 30% 2 2 Sheets Polyedrical, clear cells No Yes Yes No 0 0 0 Mucinous B Luminal A
3687 30% 2 1 Sheets, cribriform Polyedrical Yes Yes Yes No 0 0 0 Mucinous B Luminal A
3688 40% 2 2 Sheets Polyedrical, cuboidal
with apical snouts
No No No No 1+ 0 0 Mucinous B Luminal A
3690 40% 3 2 Sheets, cribriform,
rings, trabeculae,
ribbons
Polyedrical, cuboidal
with apical snouts
No No No No 0 0 0 Mucinous B Luminal A
3683 10-20% 3 2 Sheets, trabeculae Sheets, nests, cribriform Yes Yes Yes No 2+ 2+ 0 Neuroendocrine Luminal A
3695 None 3 2 Nests, sheets Spindle, hyaline No No No No 2+ 2+ 0 Neuroendocrine Luminal A
3696 None 3 2 Nests, sheets Spindle, hyaline No No No No 2+ 2+ 2+ Neuroendocrine Luminal A
3698 None 3 3 Nests, sheets Spindle, polyedrical Yes No No No 2+ 0 0 Neuroendocrine Luminal B
3699 o5% 2 2 Sheets, nests Spindle, hyaline No Yes No No 2+ 2+ 0 Neuroendocrine Luminal A
3700 None 2 1 Papillary structures,
sheets
Spindle, hyaline,
cuboidal with apical
snouts
Yes No No No 0 0 2+ Neuroendocrine Luminal A
Histological grade according to Bloom and Richardson.
Scoring of immunohistochemical staining of synaptophysin, chromogranin and CD56: 0 ¼ negative; 1+ ¼ 10–50% of tumour cells positive; 2+ ¼ 450% of tumour cells positive.
Mucinous and neuroendocrine carcinomas
1404 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 4
of neuroendocrine differentiation in 4 50% of cells
as evaluated by immunohistochemistry (Figure 2;
Table 1), whereas only 3 of the 10 mucinous A (30%)
and 2 of the 8 mucinous B tumours (25%) expressed
neuroendocrine markers in 410% of cells (Table 1).
To define the basal-like, luminal A, luminal B, HER2
and normal breast-like molecular subtype class, we
determined the correlation between the expression
Figure 1 Representative photomicrographs of mucinous A carcinoma (a) and (b), mucinous B carcinoma (c) and (d) and neuroendocrine
carcinoma (e) and (f). (a, c, e), 40 original magnification; (b, df), 400 original magnification.
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1405
Modern Pathology
(2009) 22, 14011414
Page 5
profile of each tumour with the ‘Intrinsic/UNC class
centroids described by Hu et al.
17
Mucinous cancers
were homogeneous and consistently displayed a
luminal A expression profile; neuroendocrine carci-
nomas were assigned to the luminal A (n ¼ 5) or B
(n ¼ 1) molecular subtypes (Table 1).
We next assessed whether mucinous and neu-
roendocrine carcinomas would constitute distinct
special types and discrete entities from invasive
ductal carcinomas not only at the histological but
also at the molecular level. For comparison, we
selected from a control group of 102 invasive breast
carcinomas a series of 24 invasive ductal carcinomas
that were histological grade- and molecular subtype-
matched with the cohort of mucinous and neuroen-
docrine cancers. This series consisted of 23 luminal
A and one luminal B tumours, of which 14, eight
and two were of grades 1, 2 or 3, respectively
(Supplementary Table 1). Unsupervised hierarchical
clustering using 8398 significantly regulated tran-
scripts revealed that, with the exception of three
mucinous A tumours, mucinous and neuroendo-
crine carcinomas formed a separate group. In fact,
the mucinous/neuroendocrine cluster was signifi-
cantly enriched for these tumours (Fisher’s exact test
Po10
9
; Figure 3). We further observed that in the
mucinous/neuroendocrine cluster the mucinous B
cancers were intermingled with neuroendocrine
carcinomas, whereas mucinous A tumours clustered
significantly more tightly together (Fisher’s exact
test P o10
6
). These results provide evidence to
suggest that mucinous and neuroendocrine tumours
are distinct transcriptomic entities from grade-
and molecular-subtype-matched invasive ductal
carcinomas.
Given the separation observed between muci-
nous/neuroendocrine and histological grade- and
molecular subtype-matched invasive ductal carci-
nomas in the hierarchical clustering, we sought to
define their transcriptional differences. Supervised
analysis using significance of microarray analysis
revealed 3155 transcripts differentially expressed
Figure 2 Neuroendocrine carcinoma (case 3696). Haematoxylin and eosin staining (a). Positive staining for the neuroendocrine markers
synaptophysin (b), chromogranin (c) and CD56 (d).
Mucinous and neuroendocrine carcinomas
1406 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 6
between mucinous/neuroendocrine tumours vs
grade- and molecular subtype-matched invasive
ductal carcinomas (1582 and 1573 transcripts were
preferentially expressed in mucinous/neuroendo-
crine cancers and grade- and molecular subtype-
matched invasive ductal carcinomas, respectively;
false discovery rateo0.85%; Supplementary
Table 2). Of note, extracellular matrix genes (eg,
collagens, matrix metalloproteinases, insulin-like
growth factor-binding proteins, laminins), ERBB2
and high molecular weight cytokeratins KRT5 and
KRT14 were downregulated, whereas ESR1 and the
oestrogen receptor-a (ER)-regulated genes BCL2,
ERBB4 and TFF3, the luminal KRT18 but also
CDKN1A (p21) were upregulated in mucinous/
neuroendocrine tumours compared with grade-
and molecular subtype-matched invasive ductal
carcinomas. These differences were further explored
by Ingenuity Pathway Analysis of the 3155 differ-
entially regulated transcripts identified by signifi-
cance of microarray analysis, which revealed that
extracellular matrix genes of the ‘Connective Tissue
Disorders, Dermatological Diseases and Conditions,
Genetic Disorder’ (score 36) and ‘Cell Death, Protein
Degradation, Cellular Function and Maintenance’
(score 29) networks were predominantly down-
regulated in mucinous/neuroendocrine carcinomas
compared to grade- and molecular subtype-matched
invasive ductal carcinomas (Supplementary Table 3;
Supplementary Figure 1).
To define the molecular pathways differentially
regulated between mucinous, neuroendocrine can-
cers and grade- and molecular subtype-matched
invasive ductal carcinomas, we analysed mucinous
A, mucinous B and neuroendocrine tumours sepa-
rately. Significance of microarray analysis revealed
651 transcripts differentially expressed between
mucinous A (n ¼ 10) and grade- and molecular
subtype-matched invasive ductal carcinomas
(n ¼ 10) (361 up- and 290 downregulated in muci-
nous A cancers; false discovery rate o0.85%), 597
transcripts between mucinous B (n ¼ 8) and grade-
and molecular subtype-matched invasive ductal
carcinomas (n ¼ 8) (179 up- and 418 downregulated
in mucinous B carcinomas; false discovery rate
o0.85%) and 337 transcripts between neuroendo-
crine (n ¼ 6) and grade- and molecular subtype-
matched invasive ductal carcinomas (n ¼ 6) (11 and
326 transcripts preferentially expressed in neuroen-
docrine and in grade- and molecular subtype-
matched invasive ductal carcinomas, respectively;
false discovery rate o0.85%) (Supplementary
Table 4).
Ingenuity Pathway Analysis of the 651 differen-
tially expressed transcripts identified by signifi-
cance of microarray analysis revealed four
‘Connective Tissue’-related networks (scores 39,
37, 34 and 28, respectively) among the 10 most
significant networks, in which extracellular matrix
genes (eg, collagens, laminins, matrix metallopro-
teinases) were predominantly downregulated in
mucinous A tumours compared with grade- and
molecular subtype-matched invasive ductal carci-
nomas of luminal A phenotype (Figure 4a; Supple-
mentary Figure 2a; Supplementary Table 5). Of note,
genes of the FGF family (eg, FGF10, FGF13, FGF14)
were found to be upregulated in mucinous A
cancers compared with grade-matched invasive
ductal carcinomas of luminal A subtype (Figure 4a;
Supplementary Table 4), and the differentially
expressed transcripts between mucinous A and
grade- and molecular subtype-matched invasive
ductal carcinomas were significantly enriched for
genes of the ‘FGF Signalling’ canonical pathway
IDC-NST 43
IDC-NST 68
IDC-NST 35
IDC-NST 54
IDC-NST 42
IDC-NST 66
IDC-NST 65
IDC-NST 155
IDC-NST 79
IDC-NST 179
IDC-NST 143
IDC-NST 37
Mucinous A 3680
IDC-NST 41
Mucinous A 3676
Mucinous A 3677
IDC-NST 69
IDC-NST 104
IDC-NST 122
IDC-NST 136
IDC-NST 145
IDC-NST 188
IDC-NST 157
IDC-NST 154
IDC-NST 189
IDC-NST 166
IDC-NST 87
Neuroendocrine 3698
Mucinous B 3685
Mucinous B 3686
Mucinous A 3672
Mucinous A 3671
Mucinous A 3678
Mucinous A 3673
Mucinous A 3675
Mucinous A 3674
Mucinous A 3679
Neuroendocrine 3700
Neuroendocrine 3699
Mucinous B 3684
Mucinous B 3681
Neuroendocrine 3695
Neuroendocrine 3696
Mucinous B 3682
Neuroendocrine 3683
Mucinous B 3688
Mucinous B 3687
Mucinous B 3690
Figure 3 Unsupervised hierarchical clustering of mucinous, neuroendocrine and grade- and molecular subtype-matched invasive ductal
carcinomas. Average-linkage clustering of 10 mucinous A, 8 mucinous B, 6 neuroendocrine cancers and 24 invasive ductal carcinomas of
no special type (IDC-NST) using 8398 significantly regulated transcripts.
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1407
Modern Pathology
(2009) 22, 14011414
Page 7
(P ¼ 0.0108; Supplementary Table 5). Furthermore,
the ‘Endoplasmic Reticulum Stress Response’
canonical pathway was upregulated in mucinous
A compared with grade- and molecular subtype-
matched invasive ductal carcinomas (P ¼ 0.0281;
Supplementary Figure 2b).
c
Network: Cell Morphology, Connective Tissue
Development and Function, Tissue
Development
b
Mucinous B cancers vs. grade-and
molecular subtype matched IDCs-NST
Neuroendocrinecancers vs. grade-and
molecular subtype matched IDCs-NST
Mucinous A cancers vs. grade-and
molecular subtype matched IDCs-NST
a
Network: Connective Tissue Development
and Function, Skeletal and Muscular System
Development and Function, Tissue
Development
Network: Cancer, Reproductive System
Disease, Ophthalmic Disease
Figure 4 Ingenuity Pathway Analysis. Extracellular matrix genes of the ‘Connective Tissue Development and Function, Skeletal and
Muscular System Development and Function, Tissue Development’ network (score 37) are downregulated in mucinous A vs histological
grade- and molecular subtype-matched invasive ductal carcinomas (a), of the ‘Cell Morphology, Connective Tissue Development and
Function, Tissue Development’ network (score 44) are downregulated in mucinous B vs histological grade- and molecular subtype-
matched invasive ductal carcinomas (b), of the ‘Cancer, Reproductive System Disease, Ophthalmic Disease’ (score 43) are downregulated
in neuroendocrine vs grade- and molecular subtype-matched invasive ductal carcinomas (c). Green: downregulation, red: upregulation.
IDCs-NST: invasive ductal carcinomas of no special type.
Mucinous and neuroendocrine carcinomas
1408 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 8
Ingenuity Pathway Analysis revealed that, like in
mucinous A carcinomas, downregulation of extra-
cellular matrix genes (eg, collagens, fibulins and
matrix metalloproteinases) pertaining to networks
involved in ‘Cell Morphology, Connective Tissue
Development and Function, Tissue Development’,
‘Post-Translational Modification, Cancer, Reproduc-
tive System Disease’ and ‘Cellular Movement, Skele-
tal and Muscular Disorders, Tissue Development’
(scores 44, 43 and 37, respectively) was observed in
mucinous B compared with grade-matched invasive
ductal carcinomas of luminal A phenotype (Figure 4b;
Supplementary Figure 3a; Supplementary Tables 4
and 5). Furthermore, the high molecular weight
cytokeratins KRT5 and KRT14 were downregulated,
whereas ESR1, BCL2, ERBB4 and FOXA1 were found
to be upregulated in mucinous B vs grade- and mole-
cular subtype-matched invasive ductal carcinomas
(Supplementary Table 4). Ingenuity Pathway Analysis
of genes differentially expressed between mucinous B
vs grade-matched invasive ductal carcinomas of
luminal A phenotype revealed an enrichment for
genes of the ‘p53 Signalling’ (P ¼ 0.0042) and of
the ‘Wnt/b-catenin Signalling’ canonical pathways
(P ¼ 0.0299), the latter being downregulated in muci-
nous B tumours compared with invasive ductal car-
cinomas of the same histological grade and molecular
subtype (Supplementary Table 5; Supplementary
Figure 3b).
Similar to mucinous A and B tumours, the
functional mapping of the 337 significantly differ-
entially regulated transcripts between neuroendo-
crine and grade- and molecular subtype-matched
invasive ductal carcinomas to the Ingenuity data-
base identified networks of genes having a role in
‘Cancer, Reproductive System Disease, Ophthalmic
Disease’ (score 43) and ‘Cell-To-Cell Signalling and
Interaction, Connective Tissue Development and
Function, Post-Translational Modification’ (score
42), including matrix metalloproteinases, collagens,
fibulins and ITGB1, to be downregulated in neu-
roendocrine carcinomas, as was the ‘IGF-1 Signal-
ling’ canonical pathway (P ¼ 0.0071; Figure 4c;
Supplementary Figure 4; Supplementary Table 5).
As mucinous B and neuroendocrine carcinomas
have significantly overlapping morphological fea-
tures and were intermingled in the hierarchical
clustering analysis, we defined the transcriptomic
differences of these two subtypes and histological
grade- and molecular subtype-matched invasive
ductal carcinomas. Significance of microarray ana-
lysis revealed 2315 transcripts differentially ex-
pressed (1020 up- and 1295 downregulated
transcripts in mucinous B/neuroendocrine cancers
at a false discovery rate o0.90%) between mucinous
B/neuroendocrine cancers (n ¼ 14) and grade- and
molecular subtype-matched invasive ductal carci-
nomas (n ¼ 14). (Supplementary Table 4). Of note,
transcriptional regulators (eg, MED23, GM EB1,
TCF25, EEF1A2), ESR1 and the ER-regulated
genes FOX A1, XBP1, ERBB4 and BCL2 and ‘Lipid
Metabolism, Molecular Transport, Small Molecule
Biochemistry’ gene network (score 31) were
upregulated in mucinous B/neuroendocrine carci-
nomas vs grade- and molecular subtype-matched
invasive ductal carcinomas (Supplementary
Figure 5a; Supplementary Table 5). In addition,
extracellular matrix genes (eg, collagens, laminins,
PLAU, VCAN, SERPINH1) as seen in the ‘Ophthal-
mic Disease, Cardiovascular Disease, Genetic Dis-
order’ network (score 36) were downregulated in
mucinous B/neuroendocrine carcinomas compared
with grade- and molecular subtype-matched inva-
sive ductal carcinomas (Supplementary Figure 5b).
Taken together, our results provide evidence to
suggest that mucinous A, mucinous B and neuroen-
docrine carcinomas are transcriptionally distinct
from histological grade- and molecular subtype-
matched invasive ductal carcinomas of no special
type. Furthermore, discrete molecular pathways/
networks were found to be activated in mucinous
and neuroendocrine cancers compared with grade-
and molecular subtype-matched invasive ductal
carcinomas. Most strikingly, mucinous and neu-
roendocrine tumours displayed downregulation of
extracellular matrix and connective tissue-asso-
ciated genes compared with grade- and molecular
subtype-matched invasive ductal carcinomas.
Mucinous A Cancers are Distinct Entities, Whereas
Mucinous B and Neuroendocrine Carcinomas Share
Similar Transcriptomic Profiles
Given that (i) mucinous A, mucinous B and
neuroendocrine carcinomas formed a separate clus-
ter when subjected to hierarchical clustering analy-
sis with grade- and molecular subtype-matched
invasive ductal carcinomas and that (ii) there is a
significant overlap between the morphological fea-
tures of mucinous B and neuroendocrine carcino-
mas, we sought to determine whether these
histological entities would constitute distinct enti-
ties at the transcriptomic level.
Unsupervised hierarchical clustering analysis of
mucinous A, mucinous B and neuroendocrine
carcinomas revealed that all mucinous A tumours
formed a discrete cluster, whereas mucinous B and
neuroendocrine carcinomas were intermingled in a
separate cluster (Figure 5). These results provide
evidence to suggest that mucinous A is a molecular
entity distinct from mucinous B and neuroendo-
crine carcinomas, whereas mucinous B and neu-
roendocrine carcinomas have a highly similar
transcriptome.
To further define the differences between muci-
nous A, mucinous B and neuroendocrine carcino-
mas, we performed significance of microarray
analysis using the 8398 significantly regulated
transcripts. This analysis revealed 115 transcripts
differentially expressed between mucinous A and
mucinous B carcinomas (89 up- and 26 tran-
scripts downregulated in mucinous A cancers; false
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1409
Modern Pathology
(2009) 22, 14011414
Page 9
discovery rate o1.15%; Supplementary Table 6).
Compared with mucinous B carcinomas, mucinous
A cancers were found to upregulate cell-junction
genes (eg, WTIP, GJA1), cytokeratins (eg, KRT7,
KRT23) and VIM, ITGB5 and VEGFC, but down-
regulate genes having a role in lipid synthesis/
transport (eg, AGPAT5, APOL6), as well as
ERBB4 and FOXA1 (Supplementary Table 6). The
upregulation of molecular transporters (eg,
SLC12A6), cell-junction genes as well as MET and
ITGB5 was also seen in the top network ‘Carbohy-
drate Metabolism, Lipid Metabolism, Molecular
Transport (score 39; Supplementary Table 7; Sup-
plementary Figure 6a).
The 416 differentially expressed transcripts iden-
tified between mucinous A and neuroendocrine
tumours using significance of microarray analysis
(false discovery rate o1.00%; Supplementary Table
6) showed great overlap with the 115 genes
discriminating mucinous A and mucinous B cancers
(73 genes overlap; representation factor: 12.8;
Po10
68
). For instance, downregulation of ERBB4
or FOXA1, and upregulation of VIM, FOS or IL4R
were found in both comparisons. These findings
show that the similarities in the transcriptomic
differences between mucinous A and mucinous B
and between mucinous A and neuroendocrine
carcinomas cannot be attributed by chance, provid-
ing another line of indirect evidence to suggest that
mucinous B and neuroendocrine carcinomas are
closely related at the molecular level.
Ingenuity Pathway Analysis of 416 differentially
expressed transcripts identified between mucinous
A and neuroendocrine tumours revealed ‘Cell-to-
Cell Signalling and Interaction, Tissue Develop-
ment, Cell Morphology’ (score 46) and ‘Cell
Morphology, Cellular Assembly and Organization,
Cellular Movement’ (score 41) networks of integrins
(eg, ITGA5, ITGB2, ITGB5), calcium-binding pro-
teins (eg, S100A8, S100A11) and actin cytoskeleton/
actin-binding genes (eg, DIAPH2, ANXA1, ACTN1,
AFAP, RDX, TMSB4X, VCL), which were upregu-
lated in mucinous A compared with neuroendocrine
cancers (Supplementary Figure 6b and 6c; Sup-
plementary Tables 6 and 7). These differentially
expressed transcripts were also found to be enriched
for ‘Macropinocytosis’ canonical pathway genes
(P ¼ 0.002), a clathrin-independent form of endocy-
tosis, and ‘IGF-1 Signalling’ genes (P ¼ 0.00189)
(Supplementary Figure 7; Supplementary Table 7),
which were predominantly upregulated in muci-
nous A compared with neuroendocrine cancers. Of
note, the genes identified of this ‘IGF-1 Signalling’
pathway were different from the ones of the same
pathway differentially expressed between neuroen-
docrine cancers and invasive ductal carcinomas
(Supplementary Figure 4).
Remarkably, significance of microarray analysis of
mucinous B and neuroendocrine carcinomas
showed no transcriptomic differences between these
two breast cancer types as no differentially ex-
pressed genes could be assigned (false discovery rate
o1–o52%; Supplementary Table 6). These results
were further confirmed by maxT test,
34
which failed
to show any differentially expressed transcripts
(data not shown).
Given the striking similarity between mucinous B
and neuroendocrine tumours at the molecular level,
we sought to define the transcriptomic differences
between these two histological types and mucinous
A tumours. Ingenuity Pathway Analysis of the 663
differentially expressed genes identified by signifi-
cance of microarray analysis (581 up- and 82
transcripts downregulated in mucinous A cancers,
respectively; false discovery rate o1.05%; Supple-
mentary Table 6) revealed that ‘Cell Morphology,
Mucinous A 3672
Mucinous A 3671
Mucinous A 3678
Mucinous A 3674
Mucinous A 3673
Mucinous A 3675
Mucinous A 3677
Mucinous A 3676
Mucinous A 3680
Mucinous A 3679
Neuroendocrine 3700
Neuroendocrine 3699
Mucinous B 3684
Mucinous B 3681
Neuroendocrine 3698
Mucinous B 3685
Mucinous B 3686
Neuroendocrine 3695
Neuroendocrine 3696
Mucinous B 3682
Neuroendocrine 3683
Mucinous B 3688
Mucinous B 3690
Mucinous B 3687
Figure 5 Unsupervised hierarchical clustering of mucinous and neuroendocrine carcinomas. Average-linkage clustering of 10 mucinous
A, 8 mucinous B and 6 neuroendocrine tumours using 8398 significantly regulated transcripts.
Mucinous and neuroendocrine carcinomas
1410 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 10
Skeletal and Muscular System Development and
Function, Cancer’ (score 56) and ‘Ophthalmic Dis-
ease, Cancer, Cell Signalling’ (score 48) networks of
extracellular matrix genes (eg, collagens, fibulins)
are upregulated in mucinous A compared to muci-
nous B and neuroendocrine tumours, as are TGFB1
and KRT7 (Supplementary Table 7; Supplementary
Figure 8). Furthermore, the ‘Integrin Signalling’
canonical pathway (P ¼ 0.00018) was found to be
upregulated in mucinous A vs mucinous B
and neuroendocrine carcinomas (Supplementary
Figure 9).
Taken together, our results provide evidence to
suggest that the histological entity of mucinous
carcinoma comprises two distinct molecular enti-
ties, mucinous A and mucinous B cancers, and that
mucinous B and neuroendocrine carcinomas are
strikingly similar at the transcriptomic level.
Discussion
Here, we show that by microarray-based gene
expression analysis the histological special types
mucinous and neuroendocrine carcinomas of the
breast are entities distinct from histological grade-
and molecular subtype-matched invasive ductal
carcinomas of no special type, and that different
molecular pathways are activated in these tumours.
Furthermore, our study shows that the histological
variants of mucinous carcinoma, mucinous A and
mucinous B, harbour significantly different gene
expression profiles, whereas mucinous B and
neuroendocrine carcinomas are strikingly similar
at the transcriptomic level.
Invasive ductal carcinomas were not tested for the
immunohistochemical expression of neuroendo-
crine markers. It should be noted, however, that
although 2–18% of consecutive breast cancers may
express at least focally neuroendocrine markers,
1,12
in this study neuroendocrine carcinomas formed a
separate group by genome-wide transcriptional
analysis and harboured 337 genes at a false
discovery rate o0.85% differentially expressed
when compared with grade- and molecular sub-
type-matched invasive ductal carcinomas. One
could hypothesise that if the control group com-
prised only invasive ductal carcinomas devoid of
any expression of neuroendocrine markers, even
more pronounced differences would have been
identified.
The expression of genes having a role in the
extracellular matrix and connective tissue was
significantly decreased in mucinous A, mucinous
B and neuroendocrine cancers compared to grade-
and molecular subtype-matched invasive ductal
carcinomas. This is not surprising, given that the
stroma of pure mucinous carcinomas is predomi-
nantly composed of pools of mucin with scattered
stromal cells (ie fibroblasts, endothelial and inflam-
matory cells), and the stroma of neuroendocrine
carcinomas is scant, given that these tumours are
predominantly composed of solid masses or nests
and islands of cells with few intervening stromal
cells, as opposed to luminal types of invasive ductal
carcinomas.
1,35
Another potential mechanism for
downregulation of extracellular matrix genes in
mucinous B cancers stems from the significant
downregulation of the Wnt/b-catenin canonical
signalling pathway. This pathway not only regulates
cell fate decisions, proliferation, morphology and
migration,
36
but also extracellular matrix compo-
nents and cell adhesion.
37
Our results suggest that
owing to the distinctive histological characteristics
of not only cancer cells but also stroma, mucinous
and neuroendocrine cancers may have distinct
interactions with the microenvironment or receive
distinct microenvironmental cues compared with
those of invasive ductal carcinomas.
Compared to grade-matched luminal cancers,
mucinous A and B, and neuroendocrine carcinomas
displayed higher levels of ESR1 expression, and
more overt characteristics of luminal differentiation
were observed in mucinous B and neuroendocrine
carcinomas. In fact, these tumours not only dis-
played significantly higher levels of ESR1 expres-
sion, the downstream target of ER-a activation BCL2,
the ER-pathway partners FOXA1, XBP1 and
ERBB4,
38,39
but also significant upregulation of a
network of ER-regulated lipid metabolism genes and
the luminal cytokeratin KRT18 compared to grade-
and molecular subtype-matched invasive ductal
carcinomas. Mucinous A, on the other hand, when
compared with mucinous B and neuroendocrine
carcinomas displayed decreased expression of
AKT1, FOXA1 and ERBB4. Furthermore, mucinous
A and B, and neuroendocrine cancers showed
significantly lower levels of ‘basal’ cytokeratins
KRT5 and KRT14 compared with grade- and mole-
cular subtype-matched invasive ductal carcinomas,
which is supported by earlier work showing that
CK5 and CK14 were rarely expressed in carcinomas
with neuroendocrine differentiation.
40
Our results
provide evidence to suggest that there is a spectrum
of luminal differentiation even within luminal A
cancers, and that mucinous B and neuroendocrine
tumours may have a more overt luminal phenotype
and are more homogenous at the transcriptomic
level than grade- and molecular subtype-matched
invasive ductal carcinomas.
In mucinous A tumours, we observed upregula-
tion of ESR1 but also of members of FGF family
(eg, FGF10, FGF14, FGF18) compared to grade-
matched invasive ductal carcinomas of luminal A
phenotype, and an enrichment for genes of the ‘FGF-
Signalling’ canonical pathway. Single nucleotide
polymorphisms of FGFR2 have been shown repeat-
edly to be associated with increased risk of breast
cancer, especially in ER-positive disease.
41–43
Given
that mucinous A cancers may have a consistent
activation of the FGF signalling pathway, our results
warrant further testing of FGF/ FGFR inhibitors in
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1411
Modern Pathology
(2009) 22, 14011414
Page 11
preclinical models of mucinous cancers. Ingenuity
Pathway Analysis further showed upregulation of
the canonical endoplasmic reticulum stress path-
way. The accumulation of misfolded/unfolded pro-
teins in the endoplasmic reticulum induced by
stimuli such as hypoxia or low pH has been shown
to induce a stress response (ie ‘unfolded protein
response’) and the activation of specific signalling
pathways, such as the mitogen-activated protein
kinases (MAPKs).
44–46
Epithelial mucins secreted by
breast cancer cells of mucinous tumours have been
reported to be acidic,
47,48
and we observed an
upregulation of heat shock protein genes and
MAP3K5 in mucinous A cancers compared to
grade-matched invasive ductal carcinomas of lumi-
nal A subtype. The transcriptional activation of
protein degradation and folding genes has been
described to serve as a mechanism in cancer cells in
hypoxic environments to re-establish homeostasis
and normal endoplasmic reticulum function and to
escape apoptosis induction.
49
These endoplasmic
reticulum stress response genes are currently under
investigation as potential drug targets.
49
Our results
provide a rationale for testing agents targeting
endoplasmic reticulum stress response genes in
preclinical models of mucinous A breast cancers.
We not only show here that mucinous and
neuroendocrine carcinomas of the breast are molecu-
larly distinct from histological grade- and molecular
subtype-matched invasive ductal carcinomas, but we
also provide several lines of evidence to propose that
mucinous A and mucinous B cancers as described by
Capella et al may be discrete at the transcriptomic
level,
3
whereas mucinous B and neuroendocrine
carcinomas may represent a single ‘molecular entity’
or a spectrum of closely related molecular entities.
First, hierarchical clustering analysis revealed that
mucinous A tumours form a distinct cluster from
mucinous B and neuroendocrine cancers. Second,
using significance of microarray analysis, mucinous
A and mucinous B tumours showed differential
expression of ER-regulated genes (ie ERBB4, FOXA1)
and genes of the oestrogen-regulated lipid synthesis/
transport, which indicates a reduced ER-a pathway
activation in mucinous A vs mucinous B cancers.
Third, we observed a significant overlap in genes
differentially expressed between mucinous A vs
mucinous B tumours and mucinous A vs neuroendo-
crine tumours. And finally, significance of microarray
analysis revealed no transcriptomic differences be-
tween mucinous B and neuroendocrine carcinomas.
In the seminal study by Capella et al,
3
a subgroup
of mucinous cancers (17%) displayed mixed fea-
tures of mucinous A and B cancers. Although we
have not encountered any of these tumours in this
study, one could speculate that some of these
tumours would harbour transcriptomic features of
mucinous B/neuroendocrine cancers, whereas
others would be more similar to mucinous A
tumours. Alternatively, the allocation of these
tumours to transcriptomic mucinous A or mucinous
B/neuroendocrine would potentially depend on the
percentage of each component in mucinous A/B
tumours. Finally, mucinous A/B cancers may con-
stitute yet another molecular subgroup. Further
transcriptomic analyses of mixed mucinous cancers
are warranted.
The overlapping histological features of mucinous
A and mucinous B as well as mucinous B and
neuroendocrine carcinomas are corroborated and
expanded by our transcriptomic findings. Although
we defined differences in gene expression between
mucinous A, mucinous B and neuroendocrine
cancers, these tumours were more similar to each
other than to invasive ductal carcinomas of the same
histological grade and molecular subtype, which
provides support to the contention that mucinous
and neuroendocrine carcinomas may constitute a
spectrum of differentiation. The transcriptional
homogeneity of the mucinous and neuroendocrine
cancers compared to invasive ductal carcinomas
together with the finding that mucinous cancers
have fewer genomic alterations than invasive ductal
carcinomas
50
suggest that the study of special types
of breast cancer may be an effective way of reducing
the complexity of breast cancer and expedite the
identification of biological drivers and potential
therapeutic targets for subgroups of breast cancer
patients.
2
Our transcriptome analysis of mucinous
A, mucinous B and neuroendocrine carcinomas
showed that not only basal-like breast cancers are
a heterogeneous group of tumours, but also that the
molecular subtype group of luminal A breast
cancers encompasses a diverse and heterogeneous
group of tumours in terms of gene expression,
prognosis,
51
biology and morphology.
Acknowledgements
The authors would like to thank Kay Savage for
technical assistance with the immunohistochemical
stainings. FCG and JSR-F are supported by Break-
through Breast Cancer Centre. We also acknowledge
NHS funding to the NIHR Biomedical Research
Centre.
Disclosure/conflict of interest
The authors declare no conflict of interest.
References
1 Ellis P, Schnitt SJ, Sastre-Garau X, et al. Invasive breast
carcinoma. In: Tavassoli FA, Devilee P (eds). WHO
Classification of Tumours. Pathology and Genetics of
Tumours of the Breast and Female Genital Organs.
IARC Press: Lyon, 2003, pp 9–47.
2 Reis-Filho JS, Lakhani SR. Breast cancer special types:
why bother? J Pathol 2008;216:394–398.
Mucinous and neuroendocrine carcinomas
1412 B Weigelt et al
Modern Pathology (2009) 22, 14011414
Page 12
3 Capella C, Eusebi V, Mann B, et al. Endocrine
differentiation in mucoid carcinoma of the breast.
Histopathology 1980;4:613–630.
4 Papotti M, Macrı
´
L, Finzi G, et al. Neuroendocrine
differentiation in carcinomas of the breast: a study of
51 cases. Semin Diagn Pathol 1989;6:174–188.
5 Maluf HM, Zukerberg LR, Dickersin GR, et al. Spindle-
cell argyrophilic mucin-producing carcinoma of the
breast. Histological, ultrastructural, and immunohisto-
chemical studies of two cases. Am J Surg Pathol
1991;15:677–686.
6 Papotti M, Gherardi G, Eusebi V, et al. Primary oat cell
(neuroendocrine) carcinoma of the breast. Report of
four cases. Virchows Arch A Pathol Anat Histopathol
1992;420:103–108.
7 Sapino A, Righi L, Cassoni P, et al. Expression of the
neuroendocrine phenotype in carcinomas of the breast.
Semin Diagn Pathol 2000;17:127–137.
8 Zekioglu O, Erhan Y, i1 M, et al. Neuroendocrine
differentiated carcinomas of the breast: a distinct
entity. Breast 2003;12:251–257.
9 Nassar H, Qureshi H, Volkanadsay N, et al. Clinico-
pathologic analysis of solid papillary carcinoma of the
breast and associated invasive carcinomas. Am J Surg
Pathol 2006;30:501–507.
10 Maluf HM, Koerner FC. Carcinomas of the breast with
endocrine differentiation: a review. Virchows Arch
1994;425:449–457.
11 Sapino A, Papotti M, Righi L, et al. Clinical signifi-
cance of neuroendocrine carcinoma of the breast. Ann
Oncol 2001;12:S115–S117.
12 Miremadi A, Pinder SE, Lee AH, et al. Neuroendocrine
differentiation and prognosis in breast adenocarcino-
ma. Histopathology 2002;40:215–222.
13 Makretsov N, Gilks CB, Coldman AJ, et al. Tissue
microarray analysis of neuroendocrine differentiation
and its prognostic significance in breast cancer. Hum
Pathol 2003;34:1001–1008.
14 Louwman MW, Vriezen M, van Beek MW, et al.
Uncommon breast tumors in perspective: incidence,
treatment and survival in the Netherlands. Int J Cancer
2007;121:127–135.
15 Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits
of human breast tumours. Nature 2000;406:747–752.
16 Sorlie T, Tibshirani R, Parker J, et al. Repeated
observation of breast tumor subtypes in independent
gene expression data sets. Proc Natl Acad Sci USA
2003;100:8418–8423.
17 Hu Z, Fan C, Oh D, et al. The molecular portraits of
breast tumors are conserved across microarray plat-
forms. BMC Genomics 2006;7:96.
18 Kreike B, van Kouwenhove M, Horlings H, et al. Gene
expression profiling and histopathological character-
ization of triple negative/basal-like breast carcinomas.
Breast Cancer Res 2007;9:R65.
19 Weigelt B, Horlings HM, Kreike B, et al. Refinement of
breast cancer classification by molecular characterization
of histological special types. J Pathol 2008;216:141–150.
20 Weigelt B, Kreike B, Reis-Filho JS. Metaplastic breast
carcinomas are basal-like breast cancers: a genomic
profiling analysis. Breast Cancer Res Treat 2008, e-pub
ahead of print; doi:10.1007/s10549-008-0197-9.
21 Lien HC, Hsiao YH, Lin YS, et al. Molecular signatures
of metaplastic carcinoma of the breast by large-scale
transcriptional profiling: identification of genes poten-
tially related to epithelial-mesenchymal transition.
Oncogene 2007;26:7859–7871.
22 Bertucci F, Finetti P, Cervera N, et al. Gene expression
profiling shows medullary breast cancer is a subgroup
of basal breast cancers. Cancer Res 2006;66:4636–4644.
23 Jacquemier J, Padovani L, Rabayrol L, et al.
Typical
medullary breast carcinomas have a basal/myoepithe-
lial phenotype. J Pathol 2005;207:260–268.
24 Vincent-Salomon A, Gruel N, Lucchesi C, et al.
Identification of typical medullary breast carcinoma
as a genomic sub-group of basal-like carcinomas, a
heterogeneous new molecular entity. Breast Cancer Res
2007;9:R24.
25 Azoulay S, Lae M, Freneaux P, et al. KIT is highly
expressed in adenoid cystic carcinoma of the breast, a
basal-like carcinoma associated with a favourable
outcome. Mod Pathol 2005;18:1623–1631.
26 Reis-Filho JS, Milanezi F, Steele D, et al. Metaplastic
breast carcinomas are basal-like tumours. Histopathol-
ogy 2006;49:10–21.
27 Rakha EA, Reis-Filho JS, Ellis IO. Basal-like breast
cancer: a critical review. J Clin Oncol 2008;26:
2568–2581.
28 Turner NC, Reis-Filho JS, Russell AM, et al. BRCA1
dysfunction in sporadic basal-like breast cancer.
Oncogene 2007;26:2126–2132.
29 Lo
´
pez-Bonet E, Alonso-Ruano M, Barraza G, et al.
Solid neuroendocrine breast carcinomas: incidence,
clinico-pathological features and immunohistochem-
ical profiling. Oncol Rep 2008;20:1369–1374.
30 Kreike B, Halfwerk H, Armstrong N, et al. Local
recurrence after breast conserving therapy in relation
to gene expression patterns in a large series of patients.
Clin Cancer Res 2009;15:4181–4190.
31 Moolenaar CE, Muller EJ, Schol DJ, et al. Expression of
neural cell adhesion molecule-related sialoglycopro-
tein in small cell lung cancer and neuroblastoma
cell lines H69 and CHP-212. Cancer Res 1990;50:
1102–1106.
32 Eisen M, Spellman P, Brown P, et al. Cluster analysis
and display of genome-wide expression patterns. Proc
Natl Acad Sci USA 1998;95:14863–14868.
33 Tusher VG, Tibshirani R, Chu G. Significance
analysis of microarrays applied to the ionizing
radiation response. Proc Natl Acad Sci USA 2001;98:
5116–5121.
34 Korkola JE, DeVries S, Fridlyand J, et al. Differentiation
of lobular versus ductal breast carcinomas by expression
microarray analysis. Cancer Res 2003;63:7167–7175.
35 Tomasek JJ, Gabbiani G, Hinz B, et al. Myofibroblasts
and mechano-regulation of connective tissue remodel-
ling. Nat Rev Mol Cell Biol 2002;3:349–363.
36 Brennan KR, Brown AM. Wnt proteins in mammary
development and cancer. J Mammary Gland Biol
Neoplasia 2004;9:119–131.
37 Schambony A, Kunz M, Gradl D. Cross-regulation of
Wnt signaling and cell adhesion. Differentiation
2004;72:307–318.
38 Thorat MA, Marchio C, Morimiya A, et al. Forkhead
box A1 expression in breast cancer is associated with
luminal subtype and good prognosis. J Clin Pathol
2008;61:327–332.
39 Wilson BJ, Giguere V. Meta-analysis of human cancer
microarrays reveals GATA3 is integral to the estrogen
receptor alpha pathway. Mol Cancer 2008;7:49.
40 Papotti M, Sapino A, Righi L, et al. 34betaE12
cytokeratin immunodetection in the differential diag-
nosis of neuroendocrine carcinomas of the breast. Appl
Immunohistochem Mol Morphol 2001;9:229–233.
Mucinous and neuroendocrine carcinomas
B Weigelt et al 1413
Modern Pathology
(2009) 22, 14011414
Page 13
41 Easton DF, Pooley KA, Dunning AM, et al. Genome-
wide association study identifies novel breast cancer
susceptibility loci. Nature 2007;447:1087–1093.
42 Hunter DJ, Kraft P, Jacobs KB, et al. A genome-wide
association study identifies alleles in FGFR2 asso-
ciated with risk of sporadic postmenopausal breast
cancer. Nat Genet 2007;39:870–874.
43 Garcia-Closas M, Hall P, Nevanlinna H, et al. Hetero-
geneity of breast cancer associations with five suscept-
ibility loci by clinical and pathological characteristics.
PLoS Genet 2008;4:e1000054.
44 Schro
¨
der M, Kaufman RJ. The mammalian unfolded
protein response. Annu Rev Biochem 2005;74:739–789.
45 Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum
stress: cell life and death decisions. J Clin Invest
2005;115:2656–2664.
46 Moenner M, Pluquet O, Bouchecareilh M, et al.
Integrated endoplasmic reticulum stress responses in
cancer. Cancer Res 2007;67:10631–10634.
47 Hanna WM, Corkill M. Mucins in breast carcinoma.
Hum Pathol 1988;19:11–14.
48 Sa
´
ez C, Japo
´
n MA, Poveda MA, et al. Mucinous
(colloid) adenocarcinomas secrete distinct O-acylated
forms of sialomucins: a histochemical study of gastric,
colorectal and breast adenocarcinomas. Histopathol-
ogy 2001;39:554–560.
49 Kim I, Xu W, Reed JC. Cell death and endoplasmic
reticulum stress: disease relevance and thera-
peutic opportunities. Nat Rev Drug Discov 2008;7:
1013–1030.
50 Fujii H, Anbazhagan R, Bornman DM, et al. Mucinous
cancers have fewer genomic alterations than more
common classes of breast cancer. Breast Cancer Res
Treat 2002;76:255–260.
51 Di Saverio S, Gutierrez J, Avisar E. A retrospective
review with long term follow up of 11 400 cases of pure
mucinous breast carcinoma. Breast Cancer Res Treat
2008;111:541–547.
Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/
modpathol)
Mucinous and neuroendocrine carcinomas
1414 B Weigelt et al
Modern Pathology (2009) 22, 14011414
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    • "Lacroix- Triki et al. [17] found less genetic instability, suggesting that it is not only a histological entity, but also molecularly distinct from common ductal adenocarcinoma. Others studies, especially those from the late nineties, present the neuroendocrine differentiation of these neoplasms, which can occur in a variable percentage from 21% to 42% with histochemical studies, immunohistochemistry, and ultra- strcture181920. The importance of this variety of breast adenocarcinoma is that numerous studies have shown that PMACB has a better prognosis than mixed or common ductal types [4,5]. Volkan Adsay et al. [13] propose that secretion of distinct mucin, specifically to the stromal surface, acts as a container for neoplastic cells, reducing their ability to disseminate. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Pure mucinous adenocarcinoma of the breast is a rare entity characterized by the production of variable amounts of mucin comprising 1% to 6% of breast carcinomas. Some mucinous adenocarcinomas have shown expression of intestinal differentiation markers such as MUC-2. This study examines the expression of intestinal differentiation markers in this type of breast carcinoma. Results Twenty-two cases of pure mucinous adenocarcinoma of the breast were assessed. Immunochemistry was performed for beta-catenin, CDX-2 and MUC-2. All cases were positive for B-catenin. MUC-2 positivity was observed in all cases; 63. 6% were 3 plus positive. All cases were negative for CDX-2. Conclusions These results suggest that mucinous breast carcinomas express some markers of intestinal differentiation, such as MUC-2 and beta-catenin; however, future studies with a larger series of cases and using molecular techniques that help affirm these results are needed.
    Full-text · Article · Sep 2014 · Biological research
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    • "c ana - lyses of special histologic types of breast cancer conducted by our group and others ( Bertucci et al . , 2008 ; Duprez et al . , 2012 ; Geyer et al . , 2010 ; Gruel et al . , 2010 ; Horlings et al . , 2013 ; Lacroix - Triki et al . , 2010 ; Lopez - Garcia et al . , 2010b ; Marchio et al . , 2009 , 2008 ; Vincent - Salomon et al . , 2007 ; Weigelt et al . , 2009a , 2010b , 2008 , 2009b ; Wetterskog et al . , 2012 ) have demonstrated that tumors from each of the special histologic types of breast cancer are more homogeneous amongst them - selves than IDC - NSTs . In addition , some of the histologic spe - cial types have been shown to be driven by recurrent fusion genes resultant of chromosomal t"
    [Show abstract] [Hide abstract] ABSTRACT: Integrative genomic and transcriptomic characterization of papillary carcinomas of the breast, Molecular Oncology (2014), doi: 10.1016/j.molonc.2014.06.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
    Full-text · Article · Jun 2014 · Molecular Oncology
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    • "This underlines the need to specify the subtype and grade of neuroendocrine tumor as accurately as possible. As cellular invasive mucinous carcinomas (with low-grade morphology) clusters in the luminal A molecular subgroup [7] and solid papillary carcinoma is considered to mostly represent an intraductal carcinoma with possible associated invasive carcinoma [16], these tumors obviously represent low-grade carcinomas with a very good prognosis. On the other hand small cell carcinoma of the breast is a neuroendocrine carcinoma of high-grade, although in a recent study it has been shown to be less aggressive than, for example, small cell carcinoma of the lung [9]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Carcinomas of the breast with neuroendocrine features are incorporated in the World Health Organization classification since 2003 and include well-differentiated neuroendocrine tumors, poorly differentiated neuroendocrine carcinomas/small cell carcinomas, and invasive breast carcinomas with neuroendocrine differentiation. Neuroendocrine differentiation is known to be more common in certain low-grade histologic special types and has been shown to mainly cluster to the molecular (intrinsic) luminal A subtype. Methods: We analyzed the frequency of neuroendocrine differentiation in different molecular subtypes of breast carcinomas of no histologic special type using immunohistochemical stains with specific neuroendocrine markers (chromogranin A and synaptophysin). Results: We found neuroendocrine differentiation in 20% of luminal B-like carcinomas using current WHO criteria (at least 50% of tumor cells positive for synaptophysin or chromogranin A). In contrast, no neuroendocrine differentiation was seen in luminal A-like, HER2 amplified and triple-negative carcinomas. Breast carcinomas with neuroendocrine differentiation presented with advanced stage disease and showed aggressive behavior. Conclusions: We conclude that neuroendocrine differentiation is more common than assumed in poorly differentiated luminal B-like carcinomas. Use of specific neuroendocrine markers is thus encouraged in this subtype to enhance detection of neuroendocrine differentiation and hence characterize the biological and therapeutic relevance of this finding in future studies.
    Full-text · Article · Feb 2014
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