Evidence for Transformation of Fibroadenoma of
the Breast to Malignant Phyllodes Tumor
Kurt B. Hodges, MD,* Fadi W. Abdul-Karim, MD,w Mingsheng Wang, MD,*
Antonio Lopez-Beltran, MD,z Rodolfo Montironi, MD,y Samantha Easley, MD,w
Shaobo Zhang, MD,* Nancy Wang, MD,w Gregory T. MacLennan, MD,w and Liang Cheng, MD*
Abstract: Fibroadenoma (FA) and phyllodes tumor (PT) of the
breast are biphasic tumors composed of variable proportions of
epithelial and stromal cells. We identified a case of synchronous
FA and PT in the same breast mass. Using laser capture
microdissection, loss of heterozygosity analysis was performed
on these components to gain potential insight into the
histogenetic relationship between FA and PT. Genomic DNA
was analyzed at 10 microsatellite loci by polymerase chain
reaction. Both tumors showed allelic loss at D7S522. In
addition, phylloid tumor showed allelic loss at TP53 and
D22S264, not observed in FA. Our data suggest that FA and PT
are clonally related and allelic loss at TP53 and D22S264 may be
implicated in the progression of FA to phyllode tumor.
Key Words: breast, fibroadenoma, phyllodes tumor, molecular
genetics, cancer progression, laser capture microdissection,
(Appl Immunohistochem Mol Morphol 2009;17:345–350)
variable proportions of epithelial and stromal cells. On
the basis of their histologic features, fibroepithelial
tumors have traditionally been divided into (1) simple
fibroadenoma (FA), (2) complex FA, and (3) phyllodes
tumor (PT). Simple FAs are the most common and are
characterized by loose cellular stromal connective tissue
with little nuclear pleomorphism and infrequent mitoses.
Complex FAs have additional features including calcifi-
cations, papillary apocrine metaplasia, sclerosing adeno-
sis, and/or cysts >3mm. DuPont et al1found that 22%
ibroepithelial tumors of the breast are a clinically and
pathologically diverse group of lesions composed of
of the FAs in their study had complex features. PTs are
rare, accounting for <1% of breast tumors. They are
typically more cellular and may show a spectrum of
features including stromal overgrowth, hypercellularity,
nuclear atypia, increased mitotic count, and frankly
Previous clonality studies of FA have shown
differing results. Using loss of heterozygosity (LOH),
Noguchi et al2found that both the epithelial and stromal
components were polyclonal. Similarly, Franco et al3
found no genetic abnormalities in FAs supporting the
general view that FAs are benign lesions. However,
Kasami et al4found microsatellite instability (MSI) in a
small percentage of FAs. Several other studies have also
demonstrated genetic abnormalities in FAS,5,6suggesting
that some are neoplastic. Although FAs and PTs have
traditionally been considered related, there is relatively
little molecular genetic data linking the two. Noguchi
et al6conducted clonal analysis of FA and PT that
occurred sequentially from 3 patients. They demonstrated
that the same allele of the androgen receptor gene was
inactivated in the stromal component of the tumors from
each patient suggesting that some monoclonal FAs can
progress to PTs.
This report analyzes the possible transformation of
a FA to malignant PT in a breast mass with concurrent
FA and malignant PT components. We investigated
whether microsatellite analysis may (1) document genetic
alterations in the various components of each tumor and
(2) identify genetic alterations involved in the transforma-
The patient was a 23-year-old woman with an enlarging
breast mass. An excisional biopsy was performed, which showed
a 6.0?4.0?4.0cm mass with pale, white, granular, and
lobulated cut surfaces. Small foci suggestive of hemorrhage
were noted. The histologic criteria were based on the 2003 WHO
classification of the breast and female genital organs.7Histo-
pathologic examination showed a simple FA composed of
epithelial structures surrounded by loose cellular stromal
connective tissue. Further observation showed a rather abrupt
transition to an increasingly cellular area demonstrating stromal
pleomorphism consistent with PT. The mitotic counts were
Copyrightr2009 by Lippincott Williams & Wilkins
Received for publication August 4, 2008; accepted November 12, 2008.
From the *Department of Pathology and Laboratory Medicine, Indiana
University School of Medicine, Indianapolis, IN; wDepartment
of Pathology, Case Western Reserve University, Cleveland, OH;
zDepartment of Pathology, Cordoba University, Cordoba, Spain;
and yInstitute of Pathological Anatomy and Histopathology, School
of Medicine, Polytechnic University of the Marche Region (Ancona),
United Hospitals, Ancona, Italy.
Reprints: Liang Cheng, MD, Department of Pathology and Laboratory
Medicine, Indiana University School of Medicine, 350 West 11th
Street, Clarian Pathology Laboratory Room 4010, Indianapolis,
IN 46202 (e-mail: firstname.lastname@example.org).
Appl Immunohistochem Mol Morphol?Volume 17, Number 4, July 2009www.appliedimmunohist.com|345
greater than 10 per 10 high power fields (40?) in the PT
Tissue Samples and Microdissection
Histologic sections were prepared from formalin-fixed,
paraffin-embedded tissue and were stained with hematoxylin
and eosin for microscopic examination. From these slides, the
epithelial and stromal components from both the FA and PT
were identified. The various components were microdissected
from the unstained slides using a PixCell II Laser Capture
Microdissection system (Arcturus Engineering, Mountain View,
CA) as previously described.8–13Cells from different areas of the
same component were collected and analyzed separately.
Normal tissue was microdissected from the surrounding breast
parenchyma and served as a control.
Oligonucleotide Primers and Polymerase
Ten microsatellite primer pairs were used to amplify
genomic DNA at specific loci on chromosome 7q31 (D7S522),
(D10S168, D10S571), 17p13 (TP53), 16q23.2 (D16S507), 17q
(D17S855), and 22q11.2 (D22S264). All are highly polymorphic
dinucleotide repeats. Some were chosen on the basis of
chromosome regions known to be associated with tumor
suppressor genes.14Previous studies demonstrated LOH at
these loci in a variety of tumors, including epithelial and stromal
components of PT of the prostate.15
Polymerase chain reaction (PCR) assays and gel electro-
phoresis were performed as described.16The PCR products were
run on a 6% denaturing polyacrylamide gel. The gels were dried
and autoradiography was performed at ?701C with varying
exposure times as needed. Microsatellite banding patterns at
each locus were classified as homozygous/uninformative (single
band) or heterozygous/informative (2 separate bands). The
criterion for allelic loss was complete absence of 1 allele in DNA
isolated from either the epithelial or stromal components of
both tumors compared with normal DNA.
The case was reviewed and fulfilled the diagnostic
criteria for FA and PT using the 2003 WHO classification
of the breast and female genital organs (Figs. 1, 2).7DNA
was successfully isolated from both epithelial and stromal
components of the FA and PT and surrounding normal
tissue (Fig. 2). The results of PCR amplification at the
various microsatellite loci are summarized in Table 1.
Both the FA and PT showed allelic loss in 1 or more
components. The number of specific loci lost ranged from
1 in the FA to 3 in the PT. Allelic loss on chromosome
7q31 (D7S522) was identified in the epithelial and stromal
components of both tumors (Fig. 3). In addition, both the
epithelial and stromal components of the PT showed loss
on chromosome 22q11.2 (D22S264) and 17p13 (TP53).
This study identified concordant allelic loss patterns
between the epithelial and stromal components of the FA
and corresponding components of the PT at 7q31
(D7S522) suggesting that both are clonal and neoplastic.
In addition, the PT showed additional allelic losses at
22q11.2 (D22S264) and 17p13 (TP53) suggesting these
loci may represent a primary chromosomal aberration in
the progression of FA to PT. Our proposed scheme of
events involved in the multistep malignant transformation
of FA to PT is shown in Figure 4.
FIGURE 1. Histology of synchronous fibroadenoma (FA) and phyllodes tumor (PT) in the same breast mass (A to D).
Hodges et al Appl Immunohistochem Mol Morphol ?Volume 17, Number 4, July 2009
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FAs have long been considered benign hyperplastic
lesions rather than true neoplastic processes. This view
has been supported by the observation that most FAs
behave in a benign clinical fashion and a majority
regresses with time. However, previous clonality studies
have shown differing results. There has been less
controversy regarding the neoplastic nature of PTs. These
tumors may grow rapidly and even ulcerate the overlying
skin. Recurrence rates are high in the setting of
incomplete excision, and metastasis may occur. A wide
variety of genetic abnormalities have been detected in
both the epithelial and stromal components of PT.11–13
FIGURE 2. Laser capture microdissection of FA (A to F) and malignant PT (G to L) components of a breast mass. Hematoxylin and
eosin-stained slides showing the epithelial and stromal components of the FA before and after microdissection (A and B and D and
E, respectively). G and H and J and K, respectively, show the epithelial and stromal components of the PT before and after
microdissection. FA indicates fibroadenoma; PT, phyllodes tumor.
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This is notable, considering that it is generally believed
that only the stroma can metastasize whereas the
epithelial component is benign.
Our findings are in contrast to those of previous
investigators who did not find genetic abnormalities in
FAs.2,3However, MSI and LOH were documented by
TABLE 1. Results of Loss of Heterozygosity (LOH)
ComponentsTP53 D7S522D8S133 D8S137 D8S261D10S168 D10S571D16S507 D17S855 D22S264
*The symbols 1 and 2 represent different sampled areas which were analyzed separately, concordant results were observed.
FA indicates Fibroadenoma; PT, Phyllodes tumor;
, loss of upper allele;, loss of lower allele; , both alleles present.
FIGURE 3. LOH analysis in the epithelial and stromal cell components of the FA and adjacent PT at loci D7S522, D8S261, and
TP53. Normal control tissue from same patient (N), epithelial component of the FA and PT (E1 and E2), and stromal component
of the FA and PT (S1 and S2) are shown. The symbols 1 and 2 represent different sampled areas that were analyzed separately. FA
indicates fibroadenoma; PT, phyllodes tumor; LOH, loss of heterozygosity.
Hodges et al Appl Immunohistochem Mol Morphol ?Volume 17, Number 4, July 2009
r2009 Lippincott Williams & Wilkins
McCulloch et al5who analyzed 39 FA cases at 11 loci,
and found the total incidence of MSI and LOH to be
1.0% and 1.8%, respectively. MSI and LOH were identi-
fied at D3S1514, D9S254, D13S289, THO1(11p15.5), and
SCA3(14q24.3-32.2), which were not included in our
analysis. D17S855 was the only microsatellite marker that
our 2 studies had in common, and it was found to be
negative in both cases. This suggests that locus D17S855,
which is located at the BRCA1 gene, may not play a role in
the pathogenesis of fibroepithelial tumors of the breast.
It is interesting to note that the microsatellite loci 7q31
(D7S522) and 22q11.2 (D22S264) were implicated in our
previous study of LOH in PT of the prostate gland.157q31
(D7S522) harbors a putative tumor suppressor gene
(Caveolin-1), which is frequently deleted in a wide variety
of tumors including squamous cell carcinoma of the head
and neck, adenocarcinoma of the prostate, renal cell
carcinoma, and breast cancers.17Caveolin-1 is an integral
membrane protein that is thought to function as a negative
regulator of different classes of cell signaling molecules.18
Allelic deletion of chromosome 22q is common in a
wide variety of tumors including primitive neuroectodermal
tumors and carcinomas of the breast and ovary.19–21It is
also implicated in the developmental disorders characterized
by craniofacial anomalies and conotruncal heart defects.19
There are a number of candidate tumor suppressor genes on
FIGURE 4. Schematic illustration of genomic events involved in the progression of malignant phyllodes tumor of the breast from
coexisting fibroadenoma. LOH at D7S522 occurred in both epithelial and stromal components of the breast fibroadenoma, but
TP53 gene is still intact as shown in the blue box (upper allele of D7S522 was lost in both epithelial and stromal components).
Additional allelic losses at TP53 and D22S264 may lead to progression of fibroadenoma to phyllodes tumor. E indicates epithelial
cells; S, stromal cells.
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22q including the neurofibromatosis type 2 gene that may Download full-text
be involved in the development of PT20.
TP53 is one of the most frequently mutated genes in
human cancer. The p53 protein is activated by a variety of
cellular stresses through several pathways and transacti-
vates its downstream genes, including regulators of cell
cycle, apoptosis, and DNA repair. Although the fre-
quency differs among tumor types, mutations in TP53 are
found in about 50% of human cancers. p53 protein
expression in PT of the breast have been reported in
several previous studies. Immunohistochemical analyses
have shown differential p53 expression in both the
epithelial and stromal components among benign, bor-
derline, and malignant PTs.21–24Previous molecular
studies of breast PTs have shown up-regulation of TP53
mRNA in overexpressed cases.25Although the precise
role of TP53 mutation in the malignant transformation
PT has yet to be elucidated, TP53 seems to play an
important role in the epithelial-stromal interactions in
In summary, we found that microsatellite altera-
tions are demonstrable in the epithelial and stromal
components of FA and PT of the breast, suggesting that
both components are part of the neoplastic process. In
addition, our data suggest that both components may
arise from the same clone. We found additional allelic loss
at D22S264 and TP53, suggesting that these putative
tumor suppressor genes may be implicated in the
progression of FA to PT.
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