Collagen Type III alpha1 as a useful diagnostic immunohistochemical marker
for fibroepithelial lesions of the breast

Yihong Wang MD, PhD, Murray B. Resnick MD, PhD, Shaolei Lu MD,
PhD, Yiang Hui MD, Alexander S. Brodsky PhD, Dongfang Yang, Evgeny
Yakirevich MD, Lijuan Wang MD, PhD

PII: S0046-8177(16)30169-1
DOI: doi: 10.1016/j.humpath.2016.07.017
Reference: YHUPA 3965

To appear in: Human Pathology

Received date: 9 May 2016

Revised date: 15 July 2016
Accepted date: 20 July 2016

Please cite this article as: Wang Yihong, Resnick Murray B., Lu Shaolei, Hui Yiang,
Brodsky Alexander S., Yang Dongfang, Yakirevich Evgeny, Wang Lijuan, Collagen Type
III alpha1 as a useful diagnostic immunohistochemical marker for fibroepithelial lesions
of the breast, Human Pathology (2016), doi: 10.1016/j.humpath.2016.07.017

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Collagen Type III alpha1 as a useful diagnostic immunohistochemical marker for

fibroepithelial lesions of the breast

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Yihong Wang MD, PhD*, Murray B. Resnick MD, PhD, Shaolei Lu MD, PhD, Yiang Hui MD,

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Alexander S. Brodsky PhD, Dongfang Yang, Evgeny Yakirevich MD, Lijuan Wang MD, PhD.

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Department of Pathology and Laboratory Medicine, Rhode Island Hospital and Lifespan Medical

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Center, Warren Alpert Medical School of Brown University

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*Corresponding Author:

Yihong Wang, MD, PhD

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Department of Pathology, Rhode Island Hospital and Lifespan Medical Center

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593 Eddy Street, Providence, RI 02903

Phone: 401-444-9897
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Fax: 401-444-4377
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Email: ywang6@ lifespan.org

Running Title: Collagen Type III and Breast Fibroepithelial Lesions

Disclosure: No conflict of interest to disclosure from all authors

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Summary

Phyllodes tumors of the breast constitute an uncommon group of fibroepithelial neoplasms that

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are classified into benign, borderline and malignant categories based on a constellation of

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histological characteristics including cytologic atypia, mitotic count, degree of stromal cellularity,

stromal overgrowth, and microscopic margins. Accurately and reproducibly differentiating these

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tumors is a long-standing diagnostic challenge. In addition, the distinction between benign

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phyllodes tumor (PT) from cellular fibroadenoma (FA) is especially difficult due to overlapping

microscopic features. We have previously shown differential expression of various collagens,

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including collagen type III alpha1 (Col3A) in breast carcinomas. In this study, we evaluated

clinicopathologic characteristics of 95 cases of fibroepithelial lesions including 56 PTs and 39

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FAs (25 cellular FA, 14 typical FA) and correlated them with the immunohistochemical staining
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pattern for Col3A. We found that stromal Col3A expression was significantly increased in PTs

when compared to FAs (p < 0.0001). Among the PT groups, there was significantly increased
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expression from benign tumors through borderline to malignant tumors. High Col3A expression
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was associated with PT type, irregular margin status and high mitotic activity. A distinct

periductal cuffing pattern of Col3A staining was unique to PTs and absent in FAs. These

findings suggest that Col3A can be a potential adjunct marker for both differentiating FA from

PT and assessing malignant potential in PTs.

Key words: phyllodes tumor, fibroadenoma, type III collagen, immunohistochemistry

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1. Introduction

Fibroepithelial lesions of the breast include fibroadenomas (FA) and phyllodes tumors (PT). The

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latter are less common, have potential to recur locally and may metastasize. As outlined by the

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World Health Organization (WHO) Classification of Tumors of the Breast, PTs are classified

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into benign, borderline and malignant grades based upon a spectrum of histological features such

as stromal hypercellularity, cytologic atypia, stromal overgrowth, mitotic rate, and microscopic

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margins [1]. The distinction between cellular fibroadenomas and phyllodes tumors can be

especially difficult.
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PTs present distinct challenges not only in their diagnosis, classification and predicted behavior,

but also in their clinical management. Their biologic behavior and malignant potential is difficult
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to predict based on morphologic criteria alone. While PTs are usually excised due to their greater
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tendency to recur, FAs are typically managed conservatively. Given the implications for clinical

decision-making including the decision of surgical excision and disease monitoring, it is

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important to identify pathologic biomarkers to assist in the differential diagnosis of these two

entities. Although a number of ancillary tests have been reported, a consensus regarding the

utility of these markers is lacking [2-5].

Type III collagen (Col3) is a homotrimeric fibril-forming collagen found in connective tissues

which is thought to play a role in regulating the collagen fibril diameter [6-7]. The expression of

Col3 is upregulated early in the healing process in many tissues [8-9]. Col3 has been shown to

regulate myofibroblast differentiation and scar formation after cutaneous injury [10]. We
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recently described stromal expression of various collagens including Col3 to be among the most

significantly differentially expressed transcripts in invasive ductal carcinoma that response

poorly to neoadjuvant chemotherapy. Stromal collagen expression may also serve as a prognostic

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biomarker to predict pathologic response after neoadjuvant chemotherapy [11]. Although

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fibroepithelial lesions are biphasic lesions with prominent stromal proliferation, the significance

of stromal collagen expression in their pathobiology is poorly understood. One study examined

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collagen type I and III expression in fibroepithelial lesions using second harmonic generation

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microscopy as a novel technique to quantify stromal collagen staining [12]. Data on protein

expression and its utility in the diagnosis of phyllodes tumor is lacking. In this study, we
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characterized Col3A expression patterns in fibroepithelial lesions, evaluated its role in malignant

progression of PTs, and assessed its utility in differentiating between FAs from PTs.
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2. Materials and Methods

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2.1. Cases
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With institutional review board approval, 95 cases of fibroepithelial lesions including 56 PTs

diagnosed from January 2003 to December 2015 were retrieved from the archives of the

Department of Pathology and Laboratory Medicine at the Lifespan Academic Medical Center

(Providence, Rhode Island). Forty randomly selected FAs including 25 typical and 14 cellular

FAs were included. Ten cases of normal breast tissue from reduction mammoplasties were

included for comparison. Patient demographics as well as other histological characteristics such

as tumor size and margin status were recorded. Eighty-nine of the 95 fibroepithelial lesions were
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excisional specimens. Six cases were biopsies including 5 typical FAs and 1 malignant PT. All

cases were reviewed microscopically and classified based on the WHO criteria for fibroepithelial

tumors [1] by three pathologists (Y. W., L. W. and E. Y.) and a consensus diagnosis was

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rendered. Briefly, cellular FAs were defined by prominent mild to moderately cellular stroma.

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PTs were diagnosed as benign if they met the following criteria: mild stromal hypercellularity,

mild stromal atypia, occasional mitoses (≤4 per 10 HPF), lack of stromal overgrowth, and

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circumscribed margins. Conversely, malignant PTs were diagnosed when the following features

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were present: marked stromal hypercellularity, stromal cell atypia, brisk mitotic activity (≥10 per

10 HPF), stromal overgrowth, and irregular margins. Notably, while some benign phyllodes
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tumors have a circumscribed margin, others demonstrate lobulated growth or some margin

irregularity. In contrast, fibroadenomas have smooth, well circumscribed margins. Borderline

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PTs showed some characteristics observed in malignant lesions but did not meet the consensus
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threshold for malignancy.

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2.2. Immunohistochemistry
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Collagen type III alpha1 (Col3A) expression was evaluated by immunohistochemistry. Four-

micron sections were cut from formalin-fixed paraffin-embedded tissue blocks, heated at 60°C

for 30 min, deparaffinized, and rehydrated. Antigen retrieval was performed by heating in

epitope retrieval buffer in a water bath at 95°C for 45 min. The slides were then incubated with

mouse monoclonal antibodies for 30 min at room temperature in a DAKO Autostainer

(Carpinteria, CA). Polyclonal anti- Col3A (1:250, Sigma, St. Louis, MO) was used for

immunohistochemistry. Immunoreactivity was detected using the DAKO EnVision method

according to the manufacturers recommended protocol.

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Immunohistochemistry scoring was assessed by two pathologists (Y. W. and L. W.)

independently without knowledge of the diagnosis. The stromal staining of Col3A appeared

diffuse with variable intensity. A score of the percentage of tumor cells positive, intensity of

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staining, and staining pattern was used to assess immunoreactivity. Percentage of positive tumor

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cells was scored from 0-3 (0 = 0%; 1 = <25%; 2 = 25–50%; 3 = >50%) and the intensity of

staining in tumor scored from 0-3 (0 0; 1 1+;2 2+; and 3 3+) as described previously[13].

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Periductal cuffing was noted to be present or absent. In situations where both the percentage and

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intensity scores were 3, periductal cuffing was considered present as the staining obscured the

pattern (Figure 1D and H). Only 5 malignant phyllodes tumors met the criteria. The percentage
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and intensity scores were multiplied to give a Col3A score. The Col3A scores ranged from 0-4

and were assigned as 0 (0 points), 1 (1-2 points), 2 (3-6 points), 3 (9 points). The Col3A score
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was increased by 1 if periductal cuffing was present (Figure 1). Immunohistochemical staining
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results were correlated with tumor type, grade, and other clinicopathological parameters.
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2.3 Statistical analysis

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Chi-square analysis was used to assess the immunohistochemical staining scores. Age and

mitotic counts among tumor types were compared by one-way variance analysis (ANOVA) and

Student’s t-test. Results of the immunohistochemical stains were correlated with

clinicopathological parameters such as patient’s age, tumor types, grade, and histological

features. All tests were 2-tailed with a p-value of less than 0.05 considered statistically

significant. Analyses were performed using the SAS software, JMP Pro version 11.1 (SAS, Cary,

NC, USA).
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3. Results

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3.1. Clinical information and histologic characteristics

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Patient demographics and histologic characteristics of our cases were summarized in Table 1. In

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total, 95 tumors including 39 FAs and 56 PTs included in this study. Among the FAs, 25 were

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characterized as cellular FA and 14 as typical FA. Among the 56 PTs, 37 (66.1%) were benign,

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12 (21.4%) were borderline and 7 (12.5%) were malignant. Patients with FAs were significantly

younger ranging from 13 to 59 years (mean = 30.7 years). In comparison, patients with PTs
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ranged from 19-83 years of age (mean = 46.0 (p <0.0001). Histological features such as tumor

margin (circumscribed vs. irregular) and mitotic counts were significantly different between the
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two groups (P<0.0001) (Table 1). Although statistically significant differences in tumor size

were seen, none of the typical FAs were >5cm in greatest dimension. Among the eight patients
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with tumors > 5cm were 1 cellular FA, 3 benign, 3 borderline and 1 malignant PT respectively.
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Two tumors >10 cm were PTs, one of which was benign PT and the other a borderline PT.
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Among cellular FAs and benign PTs, four out of 37 (10.8%) benign PTs had irregular borders

whereas none of the cellular FAs had irregular borders (P= 0.0378) (Table 1). There were

significant differences in patient age of presentation (p = 0.0003) and mitotic activity (p= 0.0072)

but not tumor size (p=0.5627).

3.2. Col3A expression in FAs and PTs

We evaluated 10 randomly selected whole tissue sections of normal breast from reduction

mammoplasties to determine the expression and localization of Col3A in normal breast tissue.
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Col3A expression was absent in normal breast epithelium and stroma. In fibroepithelial lesions,

Col3A expression was mostly restricted to the stroma. As shown in Figure 1A, rare scattered

stromal fibroblasts in FA showed Col3A expression. In PTs, Col3A showed increased intensity of

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staining in the stromal fibroblasts (Figure 1B - D). Expression of Col3A was significantly higher in

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PTs than FAs. All except one FA scored ≤2. In contrast, all PTs scored ≥3 with only one scoring 2

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(Table 2). In some of the fibroepithelial lesions, we observed periductal cuffing characterized by a

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condensation of Col3A immunoreactivity around epithelial ducts (Figure 1D).

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Among the different groups of PTs, all except one benign PT had an overall score ≥3. Malignant

PTs were scored 4 (p< 0.001) (Table 2). By pair-wise comparison, malignant PTs had a
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significantly higher Col3A scores than borderline tumors (p=0.018) However, no significant

differences were observed between benign and borderline PTs (p=0.1271).

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When comparing expression between cellular FAs and benign PTs, 24 out of 25 (96%) cellular

FAs had lower Col3A scores (0-2) while 35 out of 36 (97%) benign PTs had a higher Col3A score
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(3- 4) (p<0.0001) (Table 2). The pattern of periductal cuffing was universally present in all 37
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benign PTs (100%) whereas only 2 of 25 cellular FAs (8%) exhibited this pattern (p< 0.0001).

3.3. Association of Col3A score with other clinicopathologic parameters

The immunohistochemical findings were analyzed with clinicopathological parameters

summarized in Table 3. High Col3A scores were significantly associated with older age, PT type,

larger size, irregular tumor margins, higher mitotic activity and presence of periductal cuffing

pattern of Col3A staining.

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4. Discussion

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Distinction between FA and PT is clinically important. FAs behave in benign fashion while the

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biological behavior of PTs is unpredictable. The recurrence rate for benign PTs varies between

10-17%, while malignant PTs have a higher recurrence rate of 23-30% [1]. Classification of

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breast fibroepithelial lesions can be diagnostically challenging due to morphologic overlap

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between various histologic subtypes. Current classification of PTs depends upon a constellation

of histological parameters, each with two to three tiers of stratification. While many cases are
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straightforward, occasional tumors with overlapping features remain a challenge to classify as

there is no single pathognomonic histologic feature. The arbitrary and subjective nature of this
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classification system frequently leads to inter and intra-observer variations posing diagnostic
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challenges in routine clinical practice. Differentiation between cellular FA and benign PT is

particularly challenging. In a study of 21 cellular fibroepithelial lesions evaluated by 10 expert

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breast pathologists using the WHO criteria, uniform diagnostic agreement was only achieved for
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two cases [14]. Some experts have suggested that the definitive distinction between cellular FAs

and benign PTs may be unimportant given the low recurrence risk of benign PTs [15].

Prior studies have utilized immunohistochemical markers to help distinguish types of

fibroepithelial lesions. Most have focused on stromal markers such as c-kit, CD10, CD34 as well

as stromal ER and Ki-67 expression [2-5, 16]. Ki-67 and c-kit appeared helpful in differentiating

between benign and malignant PTs, but neither was useful in distinguishing between benign PT

and cellular FA. Histology remains the standard in differentiation between the latter two entities
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[14]. To clarify which microscopic and immunohistochemical parameters are more influential in

the diagnostic decision process, we analyzed these factors in our fibroepithelial lesions using the

WHO guidelines. First, cases were reviewed by two pathologists. If an agreement could not be

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reached, the case was reviewed by a third pathologist and a consensus diagnosis was rendered.

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Like prior studies, we found that age, mitotic count and tumor margins were significantly

different among the types of fibroepithelial lesions while there were no statistically significant

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differences in tumor size [1].

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We explored whether Col3A immunohistochemistry may aid in the diagnosis of difficult

fibroepithelial lesions. As a major component of the extracellular matrix, collagen is an

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important factor in tumorigenesis, local invasion, and metastasis [17]. Our prior study using gene

expression profiling analysis identified specific members of the collagen family including
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Col10A, Col3A and Col14A as potential predictive markers which correlated with pathologic
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response in breast cancer patients undergoing neoadjuvant chemotherapy [11]. A variety of

collagens are over-expressed in breast tumors contributing to their dense stromal structure [18].
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Col3 is found in many connective tissues and is expressed in early granulation tissue, playing a

role in regulating the physical properties of tissues and cutaneous wound repair. Increased

expression of Col3 in fetal tissues relative to adult tissues has led some to hypothesize that Col3

may contribute to the scarless phenotype of midgestational cutaneous wounds [10]. The effect of

Col3 expression on myofibroblast differentiation and scar formation suggests a previously

undefined role for this extracellular matrix protein in tissue regeneration and repair. We found

that Col3A expression was significantly increased in the PTs compared to FAs. Increase in

expression of Col3A from FA to PT and also from borderline PTs to PTs suggest that Col3A
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may play a key role in promoting a myofibroblast proliferation and progression of fibroepithelial

lesions in the breast. Our findings contradicted a study published by Tan et al [12] in which

positive staining was reported in FA but not in PT. One possible explanation for the discrepancy

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may be differences in the antibody clones used, which may show variable affinities. Tan et al.

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used a polyclonal antibody from Abcam (personal communication) which used a full-length

native purified protein corresponding to Collagen III aa 1-1466 as immunogen. In our study, a

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polyclonal antibody from Sigma which used collagen α-1(III) chain precursor recombinant

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protein epitope signature tag (PrEST) as the immunogen. Tissue microarrays used for staining in

the previous study. In contrast, our study utilized whole tissue sections which could have
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contributed to the differences in immunohistochemistry interpretation.
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We noted that Col3A exhibits a unique periductal cuffing staining pattern present in PT but not
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in FA. Histologically, PT stroma often exhibits “periductal condensation” which characterized

by condensed cellular stroma around clefts and ducts. However, some FAs with an
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intracanalicular pattern of growth can show periductal condensation. Interpretation of periductal

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condensation is often subjective and accordingly, is not included among diagnostic criteria for

PT. The periductal cuffing pattern highlighted by Col3A was an objective feature easily

recognized by pathologists. This unique feature may serve as a useful diagnostic adjunct to

distinguish benign PTs from FAs including those with intracanalicular growth. Identification of

this pattern would also be useful in other morphologically ambiguous fibroepithelial lesions.
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Acknowledgement

Research funding provided by Department of Pathology and Laboratory Medicine, Rhode Island

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Hospital and Lifespan Medical Center and by the Molecular Pathology Core of the COBRE

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Center for Cancer Research Development funded by the National Institute of General Medical

Sciences of the National Institutes of Health under Award Number P20GM103421.

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Figure Legend
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Figure 1 Col3A staining pattern in fibroepithelial lesions and scoring. A and B. A fibroadenoma

(FA) with scattered faint staining of stromal cells, combined score of 1 weak (intensity score of 1,
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percentage score 1) without periductal cuffing. C and D. A cellular FA with combined score of 2
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moderate (intensity stain 2, percentage stain >50%); this was a circumscribed mass with

increased mild stromal cellularity and admixed peri and intracanalicular growth pattern.
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Periductal cuffing with Col3A was absent, overall score 2. E and F. A borderline phyllodes

tumor (PT) with combined stromal score of 2 and periductal cuffing pattern present, overall

score 3. G and H. A malignant PT with strong staining, combined score of 3 (intensity 3,

percentage >50%) and periductal cuffing was considered present in this context. Original

magnification x400.
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Table 1. Clinical and histologic characteristics by tumor types

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Type Typical Cellular PT- PT- PT- p-value

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FA FA benign borderline malignant

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N= 14 25 37 12 7

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Age (Mean ± SEM) 32.9 ± 29.4 ± 41.6 ± 49.7 ± 4.8 62.6 ± 6.7 <0.0001

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2.7 2.82 1.82
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Tumor size (cm) (Mean 2.22 ± 2.94 ± 3.22 ± 4.24 ± 3.27 ± 0.1100

± SEM) 0.23 0.321 0.331 0.85 0.49

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tumor borders 14/0 25/02 33/42 7/5 1/6 <0.0001

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(circumscribed/irregular)
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Mitotic count (/10hpf) 0.21 ± 0.92 ± 1.95 ± 7 ± 1.0 14.14 ± <0.0001

(Mean ± SEM) 0.15 0.272 0.242 3.47

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1
No significant difference in tumor size between cellular FA and benign PT, p = 0.5627 (pair-wise comparison).

2
Significant difference was present between cellular FA and benign PT (pair-wise comparison) regarding age

(p=0.0003), tumor border (p=0.0378) and mitotic count (p=0.0072).

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Table 2. Col3A score in different tumor types

PT- PT- PT-

Type Typical FA Cellular FA P-value
benign2 borderline3 malignant4

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Col3A score1 P<0.0001

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0 6 (54.6%) 5 (45.5%) 0 0 0

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1 4 (30.8%) 9 (69.2%) 0 0 0

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2 4 (26.7%) 10 (66.7%)
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3 0 1 (3.1%) 26 5 (15.6%) 0

(81.3%)
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4 0 0 10 7 (29.2%) 7 (29.2%)
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(41.7%)
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Periductal cuffing P<0.0001

Absent 14 23 0 0 0
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Present 0 2 37 12 7

1
Col3A score includes Col3A staining score (0-3) and the periductal cuffing score (0-1)
2
Significant difference of Col3A scores was present between cellular FA and benign PT, p <0.0001 (pair-wise
comparison).
3
No significant difference of Col3A scores was present between benign and borderline PTs, p=0.1271 (pair-wise
comparison).
4
Significant difference of Col3A scores was present between borderline and malignant PTs, p=0.018 (pair-wise
comparison).
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Table 3. Clinicopathologic features of fibroadenoma and phyllodes tumor correlated with

Col3A score

Col3A low

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P-value

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(0-2) Col3A high (3-4)

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Age (Mean ± SEM) 31.0 ± 2.1 45.8 ± 2.0 <0.0001

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Size (cm) (Mean ±
0.0286
SEM) 2.68 ± 0.22 3.44 ± 0.29

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Mitotic count (per
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10hpf) (Mean ± <0.0001

SEM) 0.64 ± 0.19 4.57 ± 0.74

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FA vs PT <0.0001

FA 38 1
PT

PT 1 55
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Tumor margin <0.0001

Circumscribed 39 41
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Irregular 0 16

Periductal cuffing <0.0001

Absent 37 0

Present 2 56
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Figure 1