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Abstract

BACKGROUND:

Phyllodes tumor (PT) is a rare tumor showing various malignant potential. The histological grade of PT is related to clinical outcome, but its relationship between gaining of malignant potential and underlying mechanism including cancer stem cell factor was not understood yet.

OBJECTIVE:

The main purpose of this study was to determine the expression pattern of cancer stem cell marker in PT and to understand its clinical and pathological implications.

METHODS:

CD44, CD166, ALDH1, and Ki-67 immunohistochemistry were performed on a tissue microarray from 185 cases of PT specimens (138 benign, 32 borderline, 15 malignant). The immunohistochemistry result and clinicopathological parameter of each cases were compared to analyze the implications of cancer stem cell markers on PT.

RESULTS:

Borderline/malignant PT showed higher CD44 expression of the stromal component than benign PT ( $p<$ 0.001). In lower histologic grade PT, CD166 showed increased expression in the epithelial component ( $p=$ 0.019), but decreased in the stromal component ( $p<$ 0.001). Stromal overgrowth was rarely observed as the number of positive cancer stem cell markers increased in the epithelial component ( $p<$ 0.001). In the stromal component, the number of positive cancer stem cell markers was related to higher histologic grade ( $p<$ 0.001), and increased stromal cellularity ( $p<$ 0.001), stromal atypia ( $p=$ 0.003), and stromal mitosis ( $p=$ 0.002). In benign PT, CD44 negativity ( $p=$ 0.013) and a decreased number of positive cancer stem cell markers ( $p=$ 0.012) in the epithelial component were related to poor prognosis.

CONCLUSIONS:

The cancer stem cell markers, CD44 and CD166, are expressed in both the epithelial and stromal components of phyllodes tumor. Besides, ALDH1 is only expressed in stromal component. In the stromal component, expression of cancer stem cell markers increases with higher PT histologic grade. In the epithelial component, the absence of cancer stem cell marker expression is related to poor clinical prognosis.

Keywords

Breast cancer stem cell phyllodes tumor

1. Introduction

Phyllodes tumor (PT) is a rare tumor which accounts for 0.3–1.5% of all breast neoplasms and may exhibit malignant characteristics such as recurrence or metastasis [1]. Grossly, it appears as circumscribed, firm, bulging mass. Histologically, it typically exhibits prominent intracanalicular growth with leaf-like stromal projections. The epithelial component of PTs includes bland-looking luminal epithelial cell and myoepithelial cells which may be accompanied by hypercellular stromal component. PTs are histologically classified as benign, borderline, or malignant by the World Health Organization (WHO) classification [2]. The tumor shows aggressive clinical features as the histologic grade increases. There is no established regimen for metastatic phyllodes tumor, but recent studies revealed combined chemotherapy can be useful for metastatic phyllodes tumor, rather palliative radiotherapy and resection have limited roles. Thus, close follow up is required in patients with high probability of distant metastasis [3].

Cancer stem cells are defined as cells with ability of self-proliferation and differentiation in tumors, thus enabling cancer to develop, recur, and metastases. The concept of the cancer stem cell was first proposed to explain the tumorigenesis of acute myeloid leukemia (AML), in animal models [4]. The leukemic stem cells in this study expressed specific cell surface markers, such as CD34 $+$ /CD38 $-$ . Cancer stem cells and their surface markers have since been found in other cancer cell lines and common cell surface markers include CD44, CD24, CD29, CD133, epithelial specific antigen (ESA), and aldehyde dehydrogenase 1 (ALDH1) [5]. In breast carcinoma, the known cancer stem cell markers are CD44 $+$ /CD24 $-$ and ALDH1; however, cancer stem cell markers in breast PT have not been clearly identified.

Previous immunohistochemical analysis of human malignant PT and mouse xenografted human PT revealed that malignant PT possesses mesenchymal stem cell-like features such as CD44, CD166, CD90, CD177, and disialoganglioside (GD2) positivity. In addition, ALDH $+$ /GD $+$ cells from malignant PT showed neuronal differentiation indicative of stem cells in flow cytometry study [6]. The main purpose of this study was to investigate cancer stem cell marker expression, in relatively well-known markers – CD44, CD166 and ALDH1, in breast PT and its clinical implications

2. Materials and methods

2.1 Patient selection

This study was approved by the Institutional Review Board of Yonsei University Severance Hospital. PT tissue samples were obtained from patients diagnosed at the Department of Pathology of Severance Hospital from 2000 to 2010. All tissues were fixed in 10% buffered formalin and embedded in paraffin. All archival hematoxylin and eosin (H&E)-stained slides for each case were reviewed by two pathologists (JS Koo and SI Kim). The histologic grading of PT was based on the WHO Classification of Tumours of the Breast 4 ${}^{\text{th}}$ ed. [2]. Clinical factors such as patient age, recurrence of tumor, distance metastasis, disease-free survival (DFS), and overall survival (OS) are also investigated.

2.2 Tissue microarray

The representative area was selected by H&E slide review, and the corresponding area was marked on its paraffin block. The selected area was punched out with a biopsy needle, and the 5-mm tissue core was placed in a 5 $\times$ 6 recipient block. To minimize extraction bias, two tissue cores were extracted from the same paraffin block. Each tissue core was assigned a unique tissue microarray location number linked to a database that contained additional clinicopathological data.

2.3 Immunohistochemistry

The antibodies used for immunohistochemistry are listed in Table 1. Each 3- $\mu$ m formalin-fixed, paraffin-embedded tissue section was deparaffinized and rehydrated using xylene and alcohol solution, respectively. Immunohistochemical staining was performed using a Ventana Discovery XT automated stainer (Ventana Medical Systems, Tucson, AZ, USA). Antigen retrieval was performed using Cell Conditioning 1 (CC1) buffer (citrate buffer pH 6.0, Ventana Medical Systems) following the manufacturer’s instructions. Appropriate positive and negative controls were included.

Table 1
Source, clone, and dilution of antibodies used in this study

Antibody	Clone	Dilution	Company
CD44	EPR1013Y	1:100	Abcam, Cambridge, UK
CD166	EPR2759 (2)	1:100	Abcam, Cambridge, UK
ALDH1A1	EP1933Y	1:100	Abcam, Cambridge, UK
Ki-67	30-9	1:1	Roche, Basel, Switzerland

Immunohistochemical findings were assessed by light microscopy (BX53 Upright microscope, Olympus). The findings were quantified based on the proportion and intensity of stained cells. Proportions of stained cells were graded as 0: negative, 1: less than 30% positive cells, and 2: more than 30% positive cells. Intensities were graded as 0: negative; 1: weak, 2: moderate, and 3: strong. If the product of the two grades was 0–1, we diagnosed the sample as negative, and if 2–6, we diagnosed the sample as positive [7]. Ki-67 immunohistochemical staining was performed by using anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody and immunostained slides were scanned by ROCHE VENTANA iScan HT slide scanner. Stromal component of each case of phyllodes tumor was designated and Ki-67 Labeling index was calculated by Virtuoso v. 5.6.1 software. If Ki-67 labeling index of one case overs median value of all cases it was considered as high Ki-67 labeling index. In case of Ki-67 labeling index lesser than median value, it was considered as low Ki-67 labeling index. The range of number of counted cells was from 1000 to 7000 due to variable range of stromal cellularity according to histologic grade of PT.

Table 2

Clincopathologic characteristics of patients with phyllodes tumor

Parameters	Number of patients $n=$ 185 (%)	PT, benign $n=$ 138 (%)	PT, borderline $n=$ 32 (%)	PT, malignant $n=$ 15 (%)	$P$ -value
Gender					n/a
Female	185 (100)	138 (100)	32 (100)	15 (100)
Age (y, mean $\pm$ SD)	40.4 $\pm$ 12.2	39.1 $\pm$ 12.1	43.2 $\pm$ 11.0	47.6 $\pm$ 13.4	0.013
Tumor size (cm, mean $\pm$ SD)	4.0 $\pm$ 2.6	3.7 $\pm$ 2.2	4.2 $\pm$ 2.5	6.2 $\pm$ 4.3	0.001
Stromal cellularity					$<$ 0.001
Mild	107 (57.8)	105 (76.1)	2 (6.3)	0 (0.0)
Moderate	66 (35.7)	33 (23.9)	26 (81.3)	7 (46.7)
Marked	12 (6.5)	0 (0.0)	4 (12.5)	8 (53.3)
Stromal atypia					$<$ 0.001
Mild	143 (77.3)	136 (98.6)	7 (21.9)	0 (0.0)
Moderate	32 (17.3)	2 (1.4)	22 (68.8)	8 (53.3)
Marked	10 (5.4)	0 (0.0)	3 (9.4)	7 (46.7)
Stromal mitosis					$<$ 0.001
0–4/10 HPFs	142 (76.8)	138 (100.0)	4 (12.5)	0 (0.0)
5–9/10 HPFs	33 (17.8)	0 (0.0)	28 (87.5)	5 (33.3)
$\geqslant$ 10/10 HPFs	10 (5.4)	0 (0.0)	0 (0.0)	10 (66.7)
Stromal overgrowth					$<$ 0.001
Absent	169 (91.4)	138 (100.0)	29 (90.6)	2 (13.3)
Present	16 (8.6)	0 (0.0)	3 (9.4)	13 (86.7)
Tumor margin					$<$ 0.001
Circumscribed	166 (89.7)	135 (97.8)	25 (78.1)	6 (40.0)
Infiltrative	19 (10.3)	3 (2.2)	7 (21.9)	9 (60.0)
Ki-67 labeling index in stromal component	1.8 $\pm$ 6.4	0.9 $\pm$ 2.4	0.8 $\pm$ 2.8	14.8 $\pm$ 16.3	$<$ 0.001
(%, mean $\pm$ SD)
Surgical procedure					$<$ 0.001
Local excision	136 (73.5)	119 (86.2)	16 (50.0)	1 (6.7)
Wide excision	38 (20.5)	14 (10.1)	15 (46.9)	9 (60.0)
Mastectomy	11 (5.9)	5 (3.6)	1 (3.1)	5 (33.3)
Margin status					0.928
Negative	160 (86.5)	120 (87.0)	27 (84.4)	13 (86.7)
Positive	25 (13.5)	18 (13.0)	5 (15.6)	2 (13.3)
Tumor local recurrence	17 (9.2)	5 (3.6)	5 (15.6)	7 (46.7)	$<$ 0.001
Distance metastasis	7 (3.8)	0 (0.0)	0 (0.0)	7 (46.7)	$<$ 0.001
Duration of follow-up (months, median $\pm$ range)	63 (8–183)	73 (14–183)	59 (12–144)	15 (8–62)	$<$ 0.001

Figure 1.

Heat map of the expression of cancer stem cell-related proteins according to phyllodes tumor (PT) histologic grade. (red, positive, green, negative, E, epithelial component, S, stromal component).

Figure 2.

Expression of cancer stem cell-related proteins according to phyllodes tumor (PT) histologic grade. (a)–(c) H&E stain of PT cases (X200). Stromal cellularity and atypia are increased as the histologic grade increases. (d)–(f) CD44 immunohistochemical stain (X200). CD44 expression in the stromal component is higher in borderline/malignant PT than in benign PT. (g)–(i) CD166 immunohistochemical stain ((g)–(h); X200, (i); X400). CD166 expression in the epithelial component increases as the histologic grade decreases. (j)–(l) ALDH1 immunohistochemical stain ((j)–(k); X200, (l); X400). ALDH1 was only expressed in stromal component and its stromal expression is increased as the histologic grade increases. (m)–(o) Ki-67 immunohistochemical stain (X200). Ki-67 Labeling Index was high in high grade phyllodes tumor. It is associated with stromal CD166 expression.

2.4 Statistical analysis

Data were analyzed using SPSS for Windows, Version 12.0 (SPSS Inc., Chicago, IL, USA). For determination of statistical significance, Student’s $t$ and Fisher’s exact tests were used for continuous and categorical variables, respectively. $p<$ 0.05 was considered statistically significant. Time to tumor recurrence was evaluated by Kaplan-Meier survival curve and log-rank analyses. Multivariate regression analyses were performed using the Cox proportional hazards model.

3. Results

3.1 Basic characteristics of PT

The basic characteristics of the 185 patients enrolled in this study are shown in Table 2; 138 cases were diagnosed as benign, 32 were borderline, and 15 were malignant. Patient age and tumor size increased with higher histologic grades ( $p=$ 0.013 and $p=$ 0.001, respectively). Tumor recurrence and distance metastasis also increased with higher histologic grades of PT ( $p<$ 0.001 for both). All seven cases of distance metastasis were metastasis to the lung.

Table 3
Expression of cancer stem cell-related proteins in phyllodes tumor according to histologic grade ${}^{*}$

Parameters	Total $n=$ 185 (%)		PT, benign $n=$ 138 (%)		PT, borderline $n=$ 32(%)		PT, malignant $n=$ 15 (%)		$P$ -value
CD44 (E) ${}^{*}$									0.621
Negative	14	(8.2)	11	(8.0)	2	(7.4)	1	(20.0)
Positive	156	(91.8)	127	(92.0)	25	(92.6)	4	(80.0)
CD44 (S)									$<$ 0.001
Negative	78	(42.5)	69	(50.0)	4	(12.5)	5	(33.3)
Positive	107	(57.8)	69	(50.0)	28	(87.5)	10	(66.7)
CD166 (E) ${}^{*}$									0.019
Negative	27	(15.9)	19	(13.8)	5	(18.5)	3	(60.0)
Positive	143	(84.1)	119	(86.2)	22	(81.5)	2	(40.0)
CD166 (S)									$<$ 0.001
Negative	165	(89.2)	129	(93.5)	26	(81.3)	10	(66.7)
Positive	20	(10.8)	9	(6.5)	6	(18.8)	5	(33.3)
ALDH1 (E) ${}^{*}$									n/a
Negative	170	(100.0)	138	(100.0)	27	(100.0)	5	(100.0)
Positive	0	(0.0)	0	(0.0)	0	(0.0)	0	(0.0)
ALDH1 (S)									0.331
Negative	155	(83.8)	118	(85.5)	24	(75.0)	13	(86.7)
Positive	30	(16.2)	20	(14.5)	8	(25.0)	2	(13.3)

${}^{*}$ Fifteen tumors without an epithelial component were excluded. PT, phyllodes tumor, E, epithelial component, S, stromal component.

3.2 Expression of cancer stem cell-related proteins according to PT grade

The expression levels of CD44 in the stromal component ( $p<$ 0.001) and CD166 in the epithelial ( $p=$ 0.019) and stromal ( $p<$ 0.001) components were correlated with histologic grade of PT. CD44 expression in the stromal component was higher in borderline/malignant PT than in benign PT. CD166 expression in the epithelial component was higher in PT with low histologic grades, but the opposite relationship was observed in the stromal component, where CD166 expression was higher in PT with high histologic grades (Table 3, Figs 1 and 2). ALDH1 was only expressed in stromal component in all cases. There was trend between increased ALDH1 expression in stromal component and high histologic grade, but it was statistically insignificant ( $p=$ 0.331).

3.3 Expression of cancer stem cell-related proteins according to Ki-67 labeling index in stromal component

The relationship between expression pattern of cancer stem cell-related protein and Ki-67 labeling index was investigated. There was statistical significant relationship between level of stromal CD166 expression and Ki-67 labeling index value. There was high portion of CD166 positive cases in high stromal Ki-67 labeling index group. ( $p=$ 0.012, Table 4).

Table 4
Expression of cancer stem cell-related proteins in phyllodes tumor according to Ki-67 labeling index in stromal component

Parameters	Low Ki-67 LI $n=$ 109 (%)		High Ki-67 LI $n=$ 70 (%)		$p$ -value
CD44 (E) ${}^{*}$					0.540
Negative	8	(7.5)	6	(10.3)
Positive	98	(92.5)	52	(89.7)
CD44 (S)					0.877
Negative	48	(44.0)	30	(42.9)
Positive	61	(56.0)	40	(57.1)
CD166 (E) ${}^{*}$					0.719
Negative	16	(15.1)	10	(17.2)
Positive	90	(84.9)	48	(82.8)
CD166 (S)					0.012
Negative	103	(94.5)	58	(82.9)
Positive	6	(5.5)	12	(17.1)
ALDH1 (E) ${}^{*}$					n/a
Negative	106	(100.0)	58	(100.0)
Positive	0	(0.0)	0	(0.0)
ALDH1 (S)					0.057
Negative	97	(89.0)	55	(78.6)
Positive	12	(11.0)	15	(21.4)

${}^{*}$ Fifteen tumors without an epithelial component were excluded. PT, phyllodes tumor, E, epithelial component, S, stromal component.

3.4 Correlation between number of positive markers for cancer stem cell in PT and pathologic parameters

The correlation between cancer stem cell marker expression in breast PT and pathologic parameters was found. The number of positive cancer stem cell markers in the epithelial component was correlated with stromal overgrowth. As the number of positive cancer stem cell markers in the epithelial component increased, stromal overgrowth was rarely detected ( $p<$ 0.001). The cancer stem cell marker expression in stromal component showed opposite tendency; as the number of positive cancer stem cell markers in the stromal component increased, the histologic grade ( $p<$ 0.001), stromal cellularity ( $p<$ 0.001), stromal atypia ( $p=$ 0.003), and stromal mitosis ( $p=$ 0.002) increased (Fig. 2).

3.5 Impact of the expression of cancer stem cell-related proteins on patient prognosis

There was no clinical factor showing statistical significant relationship to cancer stem cell marker expression in PT (Table 5). However, in subgroup analysis, the CD44 status ( $p=$ 0.013) and stem cell marker expression in the epithelial component ( $p=$ 0.012) was correlated with DFS in benign PT. Low levels of CD44 and a decreased number of positive markers for cancer stem cells in the epithelial component were correlated with shorter DFS in benign PT (Fig. 3).

Table 5
Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis using the log-rank test

Parameters	No. of patients total/recurrence/ metastasis	Disease-free survival		Overall survival
		Median survival (95% CI) months	$P$ -value	Median survival (95% CI) months	$P$ -value
CD44 (E) ${}^{*}$			0.318		n/a
Negative	14/2/0	121 (98–144)		n/a
Positive	156/10/0	171 (164–178)		n/a
CD44 (S)			0.474		0.109
Negative	78/6/1	165 (155–175)		176 (172–181)
Positive	107/11/6	163 (152–174)		172 (163–180)
CD166 (E) ${}^{*}$			0.081		0.160
Negative	27/4/1	126 (107–145)		142 (133–152)
Positive	143/8/1	172 (165–179)		181 (178–184)
CD166 (S)			0.325		0.734
Negative	165/14/6	167 (160–175)		176 (171–181)
Positive	20/3/1	129 (102–155)		147 (133–161)
ALDH1 (E) ${}^{*}$			n/a		n/a
Negative	170/12/2	n/a		n/a
Positive	0/0/0	n/a		n/a
ALDH1 (S)			0.645		n/a
Negative	155/15/7	165 (157–173)		n/a
Positive	30/2/0	140 (126–155)		n/a

E, epithelial component, S, stromal component.

Figure 3.

Correlation between the number of positive markers for cancer stem cells in phyllodes tumor (PT) and pathological parameters. As the number of positive cancer stem cell markers in the epithelial component increases, stromal overgrowth is rarely detected. As the number of positive cancer stem cell markers in the stromal component increases, higher histologic grades, increased stromal cellularity, increased stromal atypia, and increased stromal mitosis are observed.

Figure 4.

Impact of the expression of cancer stem cell-related proteins on patient prognosis. CD44 status and number of positive markers for cancer stem cells in the epithelial component are correlated with disease-free survival (DFS) in benign phyllodes tumor (PT). CD44 negativity and decreased number of positive markers for cancer stem cells in the epithelial component are correlated with shorter DFS in benign PT.

4. Discussion

The purpose of this study was to investigate cancer stem cell marker expression in breast PT and its clinical implications. First, we found that the expression pattern of cancer stem cell markers was different in PT of different histologic grades. Previously, there was study about malignant PT possesses stem cell like feature, such as CD44, CD166, CD90, CD177 and GD2 positivity [6]. In our study, CD44 expression was elevated in the stromal component in higher histologic grade PT, an observation consistent with previous study. Second, our study revealed differences in cancer stem cell marker expression patterns between the epithelial and stromal components of PT. In high histologic grade PT, CD44 expression and ALDH1 expression in the stromal component was elevated, whereas CD166 expression in the epithelial component was decreased. Also, the correlation of CD166 expression and histologic grade is confirmed by Ki-67 labeling index. Previously, the association of increased ALDH1 expression in stromal component with high histologic grade has been studied [8]. Additionally, ALDH $+$ /GD $+$ cells are thought to be related to tumor initiation [6]. In addition to previous studies, our findings suggest that it is possible that different expression pattern of stem cell marker in stromal and epithelial component is related to the mechanism of tumor progression. Although the exact mechanism of PT development is not well understood, it is believed that epithelial-stromal interactions play an important role. In the early stage of PT development or in low histologic grade PT, cross-talk may occur between the epithelial and stromal components. In malignant transformation, the stromal component becomes independent of epithelial interaction and begins autonomous stromal growth [9, 10]. The Wnt/ $\beta$ -catenin signaling pathway is thought to be an important molecular pathway in epithelial-stromal interactions of PT [9, 10, 11]. A previous study reported that CD166-positive lung cancer stem-like cells obtain their tumorigenic and self-renewal potential via the Wnt/ $\beta$ -catenin signaling pathway [12]. Therefore, we can hypothesize that the CD166-positive epithelial component in PT may develop tumorigenic potential via the Wnt/ $\beta$ -catenin signaling pathway as a result of cross-talk with the stromal component. This hypothesis may be confirmed by further in vivo/in vitro studies.

The genes related to malignant transformation of PT include TP53, PIK3CA, RB1, NF1, PTEN, BRAF, and EGFR [13, 14, 15, 16, 17]. Interestingly, CD44-positive cancer stem cells are known to have activated EGFR signaling [18, 19, 20], TP53/PTEN mutations [21], and a hyperactive phosphatidylinositol 3-kinase pathway [22]. Therefore, it can be hypothesized that the CD44-positive stromal component in PT achieves malignant transformation by autonomous stromal growth. We propose that the CD166-positive epithelial component contributes to tumor progression in low histologic grade PT, and after gaining autonomous growth potential, the CD44-positive stromal component becomes involved in malignant transformation. Further studies are required to test this hypothesis.

In this study, the stromal overgrowth in PT increases in prominence as the number of positive markers for cancer stem cells in the epithelial component decreases. Moreover, we demonstrated that in benign PT, lower levels of CD44 and a decreased number of positive markers for cancer stem cells in the epithelial component results in shorter DFS. These findings suggest that the cancer stem cell feature of the epithelial component is lost during malignant transformation of PT. In addition, an increase in the number of positive markers for cancer stem cells in the stromal component correlates with a higher histologic grade, increased stromal cellularity, increased stromal atypia, and increased stromal mitosis. These results indicate that the cancer stem cell phenotype in the stromal component is related to tumor aggressiveness.

This study has clinical implications for the treatment of PT. Previous studies have found that CD44 [23, 24, 25], CD166 [26], and ALDH1 [27] targeted therapies were effective in suppressing cell proliferation in various cancer cell lines. We are hopeful that stem cell marker targeted therapy can also be effective in PT, although further studies are required to confirm this clinical application.

In conclusion, we have shown that the cancer stem cell markers, CD44, CD166, and ALDH1, are expressed in both the epithelial and stromal components of PT. As the histologic grade of PT increases, the predominance of cancer stem cell marker expression shifts from the epithelial component to the stromal component. Lastly, the expression of cancer cell markers in the epithelial component is correlated with the clinical prognosis in PT. The limitation of this study is on limited explanation of detailed activated molecular pathway of cancer stem cells in PT. The further studies may be required to show each molecular pathway is activated in cancer stem cells of each histologic grade and stromal/epithelial component of PT.

Footnotes

Conflict of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

References

Ben Hassouna

Damak

Gamoudi

Chargui

Khomsi

Mahjoub

Slimene

Ben Dhiab

Hechiche

Boussen

and Rahal

, Phyllodes tumors of the breast: A case series of 106 patients, Am J Surg 192 (2006), 141–147.

Lakhani

S.R.

Ellis

I.O.

Schnitt

S.J.

Tan

P.H.

and Vijver

M.J.v.d.

, World Heath Organization Classification of Tumors, IARC Press, Lyon, 2012.

Mitus

J.W.

Blecharz

Walasek

Reinfuss

Jakubowicz

and Kulpa

, Treatment of patients with distant metastases from phyllodes tumor of the breast, World J Surg 40 (2016), 323–328.

Lapidot

Sirard

Vormoor

Murdoch

Hoang

Caceres-Cortes

Minden

Paterson

Caligiuri

M.A.

and Dick

J.E.

, A cell initiating human acute myeloid leukaemia after transplantation into SCID mice, Nature 367 (1994), 645–648.

Pestell

T.G.

Lisanti

M.P.

and Pestell

R.G.

, Cancer stem cells, Int J Biochem Cell Biol 44 (2012), 2144–2151.

Lin

J.J.

Huang

C.S.

Liao

G.S.

Lien

H.C.

Hung

J.T.

Lin

R.J.

Chou

F.P.

Yeh

K.T.

and Yu

A.L.

, Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells, Breast Cancer Res 16 (2014), R29.

Won

K.Y.

Kim

G.Y.

Kim

Y.W.

Song

J.Y.

and Lim

S.J.

, Clinicopathologic correlation of beclin-1 and bcl-2 expression in human breast cancer, Hum Pathol 41 (2010), 107–112.

Zhang

Liss

A.L.

Chung

Pierce

L.J.

and Kleer

C.G.

, Stromal cells in phyllodes tumors of the breast are enriched for EZH2 and stem cell marker expression, Breast Cancer Res Treat 158 (2016), 21–28.

Sawyer

E.J.

Hanby

A.M.

Rowan

A.J.

Gillett

C.E.

Thomas

R.E.

Poulsom

Lakhani

S.R.

Ellis

I.O.

Ellis

and Tomlinson

I.P.

, The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours, J Pathol 196 (2002), 437–444.

10.

Tsang

J.Y.

Mendoza

Lam

C.C.

A.M.

Putti

T.C.

Karim

R.Z.

Scolyer

R.A.

Lee

C.S.

Tan

P.H.

and Tse

G.M.

, Involvement of alpha- and beta-catenins and E-cadherin in the development of mammary phyllodes tumours, Histopathology 61 (2012), 667–674.

11.

Sawyer

E.J.

Hanby

A.M.

Poulsom

Jeffery

Gillett

C.E.

Ellis

I.O.

Ellis

and Tomlinson

I.P.

, Beta-catenin abnormalities and associated insulin-like growth factor overexpression are important in phyllodes tumours and fibroadenomas of the breast, J Pathol 200 (2003), 627–632.

12.

Jiang

Hao

Ding

Zhang

Liu

Wei

and Xi

, The effects and mechanisms of SLC34A2 on tumorigenicity in human non-small cell lung cancer stem cells, Tumour Biol 37 (2016), 10383–10392.

13.

Karim

R.Z.

O’Toole

S.A.

Scolyer

R.A.

Cooper

C.L.

Chan

Selinger

Carmalt

Mak

Tse

G.M.

Tan

P.H.

Putti

T.C.

and Lee

C.S.

, Recent insights into the molecular pathogenesis of mammary phyllodes tumours, J Clin Pathol 66 (2013), 496–505.

14.

Lae

La Rosa

Mandel

Reyal

Hupe

Terrier

and Couturier

, Whole-genome profiling helps to classify phyllodes tumours of the breast, J Clin Pathol 69 (2016), 1081–1087.

15.

Liu

Feng

Liu

Zhang

and Niu

, C-kit overexpression correlates with KIT gene copy numbers increases in phyllodes tumors of the breast, Breast Cancer Res Treat 149 (2015), 395–401.

16.

Tan

Ong

C.K.

Lim

W.K.

C.C.

Thike

A.A.

L.M.

Rajasegaran

Myint

S.S.

Nagarajan

Thangaraju

Dey

Nasir

N.D.

Wijaya

G.C.

Lim

J.Q.

Huang

Wong

B.H.

Chan

J.Y.

McPherson

J.R.

Cutcutache

Poore

Tay

S.T.

Tan

W.J.

Putti

T.C.

Ahmad

B.S.

Iau

Chan

C.W.

Tang

A.P.

Yong

W.S.

Madhukumar

G.H.

Tan

V.K.

Wong

C.Y.

Hartman

Ong

K.W.

Tan

B.K.

Rozen

S.G.

Tan

P.H.

and Teh

B.T.

, Genomic landscapes of breast fibroepithelial tumors, Nat Genet 47 (2015), 1341–1345.

17.

Tan

W.J.

Thike

A.A.

Tan

S.Y.

Tse

G.M.

Tan

M.H.

Bay

B.H.

and Tan

P.H.

, CD117 expression in breast phyllodes tumors correlates with adverse pathologic parameters and reduced survival, Mod Pathol 28 (2015), 352–358.

18.

Liu

Wei

Qiang

Dong

and Liu

, Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer, Oncogenesis 7 (2018), 36.

19.

Ibrahim

S.A.

Gadalla

El-Ghonaimy

E.A.

Samir

Mohamed

H.T.

Hassan

Greve

El-Shinawi

Mohamed

M.M.

and Gotte

, Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways, Mol Cancer 16 (2017), 57.

20.

Wise

and Zolkiewska

, Metalloprotease-dependent activation of EGFR modulates CD44(+)/CD24(-) populations in triple negative breast cancer cells through the MEK/ERK pathway, Breast Cancer Res Treat 166 (2017), 421–433.

21.

Da Cruz Paula

Leitao

Marques

Rosa

A.M.

Santos

A.H.

Rema

de Fatima Faria

Rocha

Costa

J.L.

Lima

and Lopes

, Molecular characterization of CD44(+)/CD24(-)/Ck(+)/CD45(-) cells in benign and malignant breast lesions, Virchows Arch 470 (2017), 311–322.

22.

Hardt

Wild

Oerlecke

Hofmann

Luo

Wiencek

Kantelhardt

Vess

Smith

G.P.

Schroth

G.P.

Bosio

and Dittmer

, Highly sensitive profiling of CD44+/CD24- breast cancer stem cells by combining global mRNA amplification and next generation sequencing: Evidence for a hyperactive PI3K pathway, Cancer Lett 325 (2012), 165–174.

23.

Hibino

Shibuya

Engbring

J.A.

Mochizuki

Nomizu

and Kleinman

H.K.

, Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy, Cancer Res 64 (2004), 4810–4816.

24.

Jin

Hope

K.J.

Zhai

Smadja-Joffe

and Dick

J.E.

, Targeting of CD44 eradicates human acute myeloid leukemic stem cells, Nat Med 12 (2006), 1167–1174.

25.

Marangoni

Lecomte

Durand

de Pinieux

Decaudin

Chomienne

Smadja-Joffe

and Poupon

M.F.

, CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts, Br J Cancer 100 (2009), 918–922.

26.

Roth

Drummond

D.C.

Conrad

Hayes

M.E.

Kirpotin

D.B.

Benz

C.C.

Marks

J.D.

and Liu

, Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells, Mol Cancer Ther 6 (2007), 2737–2746.

27.

Condello

Morgan

C.A.

Nagdas

Cao

Turek

Hurley

T.D.

and Matei

, Beta-catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids, Oncogene 34 (2015), 2297–2308.

Expression of cancer stem cell markers in breast phyllodes tumor

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patient selection

2.2 Tissue microarray

2.3 Immunohistochemistry

Table 1 Source, clone, and dilution of antibodies used in this study

3. Results

3.1 Basic characteristics of PT

Table 3 Expression of cancer stem cell-related proteins in phyllodes tumor according to histologic grade *

3.3 Expression of cancer stem cell-related proteins according to Ki-67 labeling index in stromal component

Table 4 Expression of cancer stem cell-related proteins in phyllodes tumor according to Ki-67 labeling index in stromal component

3.5 Impact of the expression of cancer stem cell-related proteins on patient prognosis

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis using the log-rank test

Footnotes

Conflict of interest

References

Table 1
Source, clone, and dilution of antibodies used in this study

Table 3
Expression of cancer stem cell-related proteins in phyllodes tumor according to histologic grade ${}^{*}$

Table 4
Expression of cancer stem cell-related proteins in phyllodes tumor according to Ki-67 labeling index in stromal component

Table 5
Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis using the log-rank test