J Mol Hist (2014) 45:35–45 DOI 10.1007/s10735-013-9523-6
ORIGINAL PAPER
CD44/CD24 as potential prognostic markers in node-positive invasive ductal breast cancer patients treated with adjuvant chemotherapy Agnieszka Adamczyk • Joanna A. Niemiec • Aleksandra Ambicka • Anna Mucha-Małecka Jerzy Mitus´ • Janusz Rys´
•
Received: 23 April 2013 / Accepted: 1 July 2013 / Published online: 9 July 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The hypothesis on cancer stem cells assumes the existence of small subpopulation of cells that possess the ability to undergo self-renewal and can give rise to the diversity of differentiated cells that form the tumour. It has been accepted that CD44?/CD24-/low phenotype is one of the features characterizing breast cancer stem cells. The aim of our study was to assess (1) prognostic significance of CD44/CD24 expression as well as (2) a relation between the above-mentioned phenotype and breast cancer subtypes [based on estrogen (ER), progesterone receptors, human epidermal growth factor receptor 2 and Ki67 status] and expression of selected markers such as fascin, laminin-5 gamma-2 chain, cytokeratin (CK) 5/6 and 8/18, epidermal growth factor receptor (EGFR), smooth muscle actin, P-cadherin and lymphocytic infiltration in invasive ductal breast cancer patients (T C 1, N C 1, M0), who underwent mastectomy followed by chemotherapy (with taxanes
and/or anthracyclines) or/and hormonotherapy. We noted that most cancers with CD44-/CD24- and CD44-/ CD24? phenotype were ER positive. The majority of CD44-/CD24-, CD44-/CD24? and CD44?/CD24tumours were characterized by CK5/6 and EGFR negativity. In univariate analysis we demonstrated that patients with pN1/pN2 and with CD44 ?/CD24- carcinomas had significantly lower risk of progression or cancer-related death than those with pN3 or tumours characterised by other CD44/CD24 expression patterns. We also found 100 % DFS in 12 patients with CD44?/CD24-/CK5/6?/ ER- phenotype. Other analysed parameters were insignificant. We conclude that tumours with immunophenotypes: CD44?/CD24- and CD44?/CD24-/CK5/6?/ERmight be more sensitive for chemotherapy based on taxanes and/or anthracyclines. Keywords Breast cancer CD24 CD44 Immunophenotype Disease-free survival
A. Adamczyk (&) J. A. Niemiec Department of Applied Radiobiology, Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Cracow Branch, Garncarska 11, 31-115 Krako´w, Poland e-mail:
[email protected] A. Ambicka J. Rys´ Department of Tumour Pathology, Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Cracow Branch, Krako´w, Poland A. Mucha-Małecka Department of Head and Neck Cancers, Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Cracow Branch, Krako´w, Poland J. Mitus´ Department of Surgical Oncology, Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Cracow Branch, Krako´w, Poland
Introduction Breast cancer is one of the main causes of death in women. It is well known that this neoplasm is a heterogeneous entity and encompasses several histological patterns and molecular subtypes including: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpressing (HER2?), and triple negative (TNP) (Sorlie et al. 2003). Sensitivity of the above-mentioned subtypes to anthracyclines and/or taxanes is discussed (Konecny et al. 2010; Harris et al. 2006; Rouzier et al. 2005; Hugh et al. 2009; Fountzilas et al. 2012). However, according to St. Gallen 2011 recommendations anthracyclines and taxanes should be included in chemotherapy regimens in luminal B
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subtype as well as in HER2? and TNP carcinomas (Goldhirsch et al. 2011). The hypothesis on cancer stem cells assumes the existence of small subpopulation of cells that possess the ability to undergo self-renewal and can give rise to the diversity of differentiated cells that form the tumour (Ailles and Weissman 2007; Oliveira et al. 2010). Al-Hajj et al. (2003) showed that small number of immunoselected CD44?/CD24-/low breast cancer cells, after a series of passages, had an ability to form tumours in nude mice. Hence, it is suggested that the above-mentioned phenotype is one of the features of breast cancer stem cell (Oliveira et al. 2010; Badve and Nakshatri 2012). On the other hand, Dontu et al. (2004) proposed a model of carcinogenesis in which basal tumours arise from undifferentiated estrogennegative progenitor or stem cells, while Bocker et al. (2002) suggested their origin from poorly differentiated progenitor cells expressing cytokeratin (CK) 8/18 and CK5/6 or from CK8/18-/CK5/6? stem cells. Studies aimed at determining tumour stem cells phenotype are ongoing. The origin of basal-like carcinomas from early progenitor cells could be proven by identification of the following histological/immunophenotypic features: estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) negativity, CK5/6 positivity (Nielsen et al. 2004; Paredes et al. 2007; Thike et al. 2010; Tischkowitz et al. 2007), poor differentiation, high proliferation index (Anders and Carey 2009; Rakha et al. 2006) and higher percentage of CD44?/CD24- cells (Honeth et al. 2008). The prognostic significance of CD44?/CD24- phenotype is recently meticulously investigated, although the results remain controversial (Honeth et al. 2008; Abraham et al. 2005; Athanassiadou et al. 2009; Horiguchi et al. 2010; Idowu et al. 2012; Kim et al. 2011; Ricardo et al. 2011; Sung et al. 2010; Surowiak et al. 2006; Tanei et al. 2009), what might be caused by differences in clinical/histological characteristics of studied cases, treatment schedules applied or methods used for protein evaluation. It is also possible that parameters associated with tumour stroma remodelling (lymphocytic infiltration or expression of particular extracellular matrix components), or associated with tumour cell motility (fascin expression) as well as expression of basal markers (CK5/6, epidermal growth factor receptor [EGFR], P-cadherin, smooth muscle actin [SMA]), adhesion molecules (epithelial cell adhesion molecule Ep-CAM), or aberrant CK8/18 expression might help the CD44 ?/CD24- cells to form metastases or recurrent tumours. Another factor that may have an impact on tumour recurrence is the surgical margin status, due to molecular changes in cells surrounding the tumour (Jia et al. 2012).
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Fascin is a member of the actin cytoskeletal family of proteins and plays an important role in cell motility and invasiveness of breast cancer cells (Al-Alwan et al. 2011; Yoder et al. 2005). Moreover, it is suggested that laminin-5 is related to the aggressiveness of tumour cells (Catusse et al. 2000; Skyldberg et al. 1999). The above-presented arguments prompted us to: (1) investigate a relation between CD24/CD44 phenotype and tumour stroma characteristics (lymphocytic infiltration), parameters associated with tumour cell motility and invasiveness (fascin, laminin-5 expression) or expression of basal markers and adhesion molecules and (2) evaluate the prognostic significance of the above-mentioned parameters in a group of T C 1, N C 1, M0 invasive ductal breast cancer patients treated with chemotherapy based on anthracyclines and/or taxanes.
Materials and methods Patients The studied group consisted of 156 invasive ductal breast cancer patients with metastases in lymph nodes but without distant metastasis (T C 1, N C 1, M0), who underwent mastectomy (at the Department of Surgical Oncology, Centre of Oncology, Cracow Branch), followed by chemoor/and hormonotherapy. The patients were selected consecutively according to the above-mentioned criteria between 2001 and 2011. An additional criterion was the amount and quality of tissue in paraffin blocks. The mean age of patients was 55.8 ± 0.9 (SE) (range 24.0–84.0) years. Survival analysis was conducted in a subgroup of 102 patients, who were followed-up longer than 6 months and treated with chemotherapy based on anthracyclines and/or taxanes. Only 2 patients from the investigated group received trastuzumab. Patients included into the study received treatment between 2001 and 2005, while in Europe trastuzumab was recommended for patients with HER2 overexpression from 2006 (Goldhirsch et al. 2007). Clinical and histological parameters of this group are summarized in Table 1. The study has gained approval from the Local Ethics Committee (ethics committee approval number: 11KBL/OIL/2009 and 12KBL/OIL/2009). Material Archival tumour specimens were obtained from the Department of Tumour Pathology and reviewed by two pathologists (J.R., A.A.) independently to confirm histological diagnosis and tumour grade.
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Table 1 Clinicopathological characteristics of 102 ductal breast cancer patients for whom analysis of survival was conducted Parameter pT
N (%) 1
15 (14.7)
2
83 (81.4)
C3 pN
a
Grade
Mastectomy
63 (61.7)
2
22 (21.6)
3
17 (16.7)
1 2
13 (13.0) 33 (33.0)
3
54 (54.0)
Patey/Madden
99 (97.1)
Halsted Chemotherapy
Hormonal therapy
3 (2.9)
AC
12 (11.8)
FAC
17 (16.7)
TAC
25 (24.5)
AC-T
40 (39.2)
TC
7 (6.8)
T
1 (1.0)
Not administered
31 (30.4)
Tamoxifen
40 (39.2)
Tamoxifen ? aromatase inhibitor
27 (26.5)
Tamoxifen ? GnRH analogue Trastuzumab
4 (3.9)
1
Aromatase inhibitor Not administered Administered
2 (2.0) 2 (2.0) 100 (98.0) 2 (2.0)
FAC: 5-fluorouracil, doxorubicin, cyclophosphamide—2 patients (11.8 %) received 4 cycles; 15 patients (88.2 %) received 6 cycles, AC: doxorubicin and cyclophosphamide—1 patient (8.3 %) received 2 cycles; 2 patients (16.7 %) received 3 cycles; 1 patient (8.3 %)—4 cycles; 5 patients (41.7 %)—5 cycles; while 3 patients (25.0 %) received 6 cycles, AC-T: AC—4 cycles, paclitaxel—8 cycles, TAC: docetaxel, doxorubicin, and cyclophosphamide—6 cycles, TC: docetaxel and cyclophosphamide—6 cycles—7 patients, T: docetaxel—8 cycles a
Grade was not assessed in two cases
Immunohistochemistry Sections (4 lm), prepared from tissues fixed in 10 % neutral buffered formalin and embedded in paraffin, were mounted on SuperFrostÒ Plus slides (Menzel-Gla¨ser, Braunschweig, Germany), deparaffinised and hydrated through a series of xylenes and alcohols. Following antigen retrieval (Table 2), slides were incubated in 3 % H2O2 diluted in methanol for 30 min. to block the activity of endogenous peroxidases. Non-specific binding of antibodies was blocked with 2.5 % horse normal serum (20 min.). Next, slides were incubated overnight with primary antibody at 4 °C (for P-cadherin it was only 1 h incubation at 37 °C). Studied proteins were visualized with BrightVision detection system (Immunologic, Duiven,
The Netherlands) and 3,30 -diaminobenzidine (DAB) (Vector Laboratories, Inc., Burlingame, USA). Hematoxylin was used for nuclear counterstaining. The details on the immunohistochemical (IHC) stainings, together with the number of stained cases and number of slides with positive staining, are shown in Table 2. We did not obtain IHC results for all slides. The lack of results for some markers is the effect of a small amount of tissue in paraffin blocks or small fragments of tumour tissue that hindered obtaining reliable results. Moreover, PR/HER2, EGFR/Ep-CAM and CD4/CD8 were visualised using double-staining procedure. Progesterone receptor, EGFR and CD8 were detected with VIP (Vector Laboratories, Inc., Burlingame, USA) as peroxidise substrate, while HER2, Ep-CAM and CD4 using DAB. Eventually, slides were counterstained with Methyl Green (Vector Laboratories, Inc., Burlingame, USA).
IHC evaluation IHC stainings were evaluated in the invasive component of the tumours, only. ERa and PR expression were considered positive if [1 % of tumour cells showed nuclear immunopositivity. Only tumours with complete intensive (3?) membranous HER2 staining of [30 % of cells were considered positive, according to ASCO recommendation (Wolff et al. 2007). The same scale was used for Ep-CAM evaluation. P-cadherin immunopositivity was defined as complete strong membranous staining observed in [10 % of cells or strong cytoplasmic staining in [50 % of cells. Expression of CK5/6 and CK5, EGFR, SMA, fascin, laminin-5 was considered positive if more than 1 % of tumour cells were found to be immunoreactive. Non-specific, aberrant, membranous expression of CK8/18 was regarded as basal marker. Evaluation of CD24 and CD44 expression was conducted separately, with each protein visualised on different slide. Expression was considered positive if [10 % of tumour cells showed cytoplasmic and/or membranous immunopositivity, respectively (Fig. 1a, b) (adapted from Kim et al. 2011). On the basis of CD24 and CD44 (assessed separately), four tumour immunophenotypes were distinguished: tumours in which neither CD44 nor CD24 expression was detected (CD44-/CD24-), tumours with expression of one marker (CD44?/CD24-, CD44-/ CD24?) and tumours expressing both proteins (CD44?/ CD24?). Based on CD4/CD8 expression, three different patterns of lymphocytic infiltration were identified: 0—absent or weak infiltration, TS—mild, moderate or strong lymphocytic infiltration in tumour stroma, TC—intensive lymphocytic infiltration dispersed among tumour cells.
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Table 2 Details on immunohistochemical staining Antigen
Clone
Manufacturer
ERa
6F11
Leica Biosystemsa a
Antigen retrieval
Dilution
POD substrate
Number of stained/ positively stained cases
TRS, pH = 6.1 DAKOb, 50 min., 96 °C
1:100
DAB
150/105
PR
PGR/2
Leica Biosystems
1:200
VIP
147/75
HER2
–
DAKOb
1:250
DAB
151/26
Ki 67
MIB-1
DAKOb
1:75
DAB
137/137
CK5/6, CK5
D5/16 B4, XM26
DAKOb Thermoc
1:50, 1:80
DAB
143/34
P-cadherin
56
BDd
1:200
DAB
136/66
Fascin CD4
55 k-2 SP35
CellMarquee CellMarquee
1:200 1:75
DAB DAB
133/37 144/144
CD8
C8/144B
CellMarquee
1:100
VIP
144/144
CD44
156-3C11
Thermoc
1:2000
DAB
144/70
CD24
SN3b
Thermoc
1:75
DAB
146/28
Laminin-5
4G1
DAKOb
TRS, pH = 9 DAKOb, 50 min., 96 °C
1:50
DAB
133/54
SMA
asm-1
Leica Biosystemsa
–
1:50
DAB
144/19
EGFR
H11
DAKOb
Proteinase Kb, 10 min., 37 °C
1:200
VIP
135/15
Ep-CAM
VU-1D9
Leica Biosystemsa
1:50
DAB
135/127
CK8/18
5D3
Leica Biosystemsa
1:200
DAB
152/33
a
Leica Biosystems Newcastle Ltd, Newcastle, UK
b
DakoCytomation Denmark A/S, Glostrup, Denmark
c
Thermo, Fisher Scientific, Fremont, CA, USA
d
BD Biosciences Pharmingen, BD Transduction LaboratoriesTM, Franklin Lakes, NJ, USA
e
Cell Marque, Rocklin, USA
Fig. 1 Expression of: a CD24 (cytoplasmic) and b CD44 (membranous) in breast cancer cells
MIB-1 labelling index (MIB-1 LI) was calculated as the percentage of Ki-67 immunopositive cells. Between 500 and 1,000 cells (at 9400 magnification) were counted in 5–10 fields for each slide. MIB-1 LI was assessed by two biologists independently. Strong correlation between the two measures (p = 0.000, r = 0.688) was found. Eventually, mean value of the two measures was calculated. On the basis of ER, PR, HER2 status and MIB-1 LI, four immunophenotypes were distinguished according to St. Gallen 2011 criteria (Goldhirsch et al. 2011): (1) luminal A
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(LA): ER? and PR? and HER2- and MIB-1 LI B 14 %, (2) luminal B (LB): ER? and PR? and HER2- and MIB-1 LI [ 14 % or ER? and PR? and HER2?, (3) HER2 overexpressing (HER2?): ER- and PR- and HER2? (4) triple negative phenotype (TNP): ER- and PR- and HER2-. Statistical analysis Descriptive statistics were used for the determination of mean values and standard errors of means (SE). Statistical
J Mol Hist (2014) 45:35–45
significance of differences between the frequency of events distribution in the investigated categorical variables (grade, subtype, and markers expression) were found using Pearson v2 test (Chi square test for independence). Disease-free survival (DFS) was defined as the time from surgery to the clinical or radiographic relapse of the tumour and overall survival (OS) was defined as time from surgery to death. The probability of disease-free survival and overall survival was determined using the Kaplan–Meier method. The log-rank test was used to investigate the statistical significance of the differences observed between groups. All variables related to survival in the univariate analysis were entered into Cox proportional hazard model. In all statistical procedures, p \ 0.05 was considered significant. STATISTICA 9 software, (StatSoft, Inc., Tulsa, OK, USA) was used for calculations.
Results Relations between CD24, CD44, fascin, and breast cancer subtype, basal markers and adhesion molecules The studied group consisted of 9 (6.5 %) luminal A, 85 (61.1 %) luminal B, 10 (7.2 %) HER2 overexpressing and 35 (25.2 %) triple negative cancers (classification including MIB-1 LI according to St. Gallen 2011 recommendations). In the analysed group CD44 and CD24 immunopositivity was noted in 70/144 (48.6 %) and 28/146 (19.2 %) cases, respectively. We observed fascin expression in tumour cells in 37/133 (27.8 %) cases. The lack of CD24 expression was significantly related to EGFR immunonegativity. However, the lack of CD44 was observed more frequently in LA and LB subtypes, ER? tumours, and tumours without expression of CK5/6, EGFR, SMA, P-cadherin (Table 3). With respect to CD44 and CD24 expression, four subgroups were indicated: CD44?/CD24-, CD44?/CD24?, CD44-/CD24?, and CD44-/CD24?. Most cancers with CD44-/CD24- and CD44-/CD24? phenotype were ER positive. The majority of tumours with CD44-/CD24-, CD44-/CD24? and CD44?/CD24- phenotype lacked CK5/6 and EGFR expression (Table 3). Tumours without fascin expression more frequently were: ER and PR positive, low grade (G1 or G2), LA or LB, basal markers (CK5/6, CK8/18, EGFR, SMA) and P-cadherin immunonegative, and with lymphocytic infiltration in tumour stroma (Table 3). Survival analysis Survival analysis was conducted in a subgroup of 102 patients, who were followed-up longer than 6 months and
39
treated with chemotherapy based on anthracyclines and/or taxanes. Forty-five percent of patients were followed-up for [96 months, only for 15 % patients follow-up was shorter than 36 months. The cumulative 10-year disease-free survival probability estimated using Kaplan–Meier method was 75 % (24 complete/78 censored). During follow-up 17 deaths occurred in the investigated group of 102 patients. The cumulative 10-year overall survival probability estimated using Kaplan–Meier method was 82 % (17 complete/85 censored). Mean time of follow-up was 83 months (range 10–131). Lymph node status statistically significantly influenced survival of breast cancer patients (Table 4). It is not surprising, as node involvement is a well known clinical prognostic and predictive parameter. Based on CD24 or CD44 expression only, we were not able to predict survival (Table 4). However, patients with tumour characterised by CD44 positivity and CD24 negativity (CD44?/CD24-) survived significantly longer (DFS and OS) than patients having tumours with other phenotypes (CD44?/CD24?, CD44-/CD24?, CD44-CD24-) (Fig. 2a, Table 4). Moreover, we analysed DFS and OS according to studied parameters in subgroups with particular chemotherapy schedule: (1) anthracyclines, (2) anthracyclines and taxanes, (3) taxanes. In the subgroup treated with anthracyclines and taxanes significantly longer survival was noted for patients with CD44? (DFS p = 0.019, OS p = 0.062) and CD44?/CD24- (DFS p = 0.006, OS p = 0.019) tumours. In the subgroups of patients treated only with anthracyclines or taxanes none of analysed parameters reached statistical significance, probably due to the relatively small number of patients (29 patients treated with anthracyclines and 8 with taxanes). Finally, we have studied the survival of patients according to the cumulative effect of potential markers of breast cancer stem cell (CD44, CD24, ER, and CK5/6 expression). We noted 100 % survival in 12 patients with tumours characterized by expression of CD44 and CK5/6 and lack of expression of CD24 and ER (CD44?/CD24-/ CK5/6?/ER- phenotype) (Fig. 2b) (DFS p = 0.046, OS p = 0.112). None of the 12 patients from the above-mentioned group received hormonotherapy or trastuzumab, while chemotherapy was applied to all patients (4 received anthracyclines, 1—taxanes, while 7—both anthracyclines and taxanes). Among CD44-/CD24?/ER-/CK5/6? tumours most were pT2 (only one was pT1) and G3 (only one was G2). Our results, that suggest the lack of risk of breast cancer progression in patients with CD44-/CD24?/ ER-/CK5/6? tumours, should be confirmed in a larger cohort. None of the other studied parameters (tumour size [mostly pT2], grade, chemotherapy/hormonotherapy schedule, breast cancer subtype according to St. Gallen 2011,
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123 62 3 65 5 6 40 17 36 29 2 58 10 42 23 51 15
TC 0 1 0 1
38 62 12 6 47 3 11 61 10 58 14
1 0 1 LA LB HER2? TNP 0 1 0 1
0 1 0 1 0 1 2 0 1 0 TS
7 30 36 15 57 34
1 2 3 0 1 0
(7.3 %) (32.8 %) (18.0 %) (40.2 %) (11.8 %)
(48.1 %) (2.3 %) (47.4 %) (3.6 %) (4.6 %) (31.0 %) (13.2 %) (27.9 %) (22.5 %) (1.5 %) (42.6 %)
(27.3 %) (44.0 %) (8.5 %) (4.6 %) (35.9 %) (2.3 %) (8.4 %) (44.2 %) (7.2 %) (40.8 %) (9.9 %)
(4.9 %) (21.1 %) (25.4 %) (10.6 %) (40.1 %) (24.5 %)
3 32 31 40 21
53 11 53 14 1 46 19 29 35 7 56
32 54 13 3 33 7 21 44 23 53 17
9 20 40 28 42 35
98 9 97 14 8 70 28 55 52 8 94 11 62 42 75 28
(2.2 %)l (25.0 %) (24.2 %) (31.5 %) (16.5 %)
60 96 19 8 68 6 26 88 25 91 26
14 45 57 32 83 53
(41.1 %) (8.5 %)h (38.7 %) (10.3 %)k (0.9 %) (35.6 %) (14.7 %) (22.5 %) (27.1 %)l (5.2 %) (41.2 %)
(23.0 %) (38.3 %) (9.2 %) (2.3 %) (25.2 %) (5.3 %) (16.0 %)d (31.9 %) (16.7 %)e (37.3 %) (12.0 %)
(6.3 %) (14.1 %) (28.2 %) (19.7 %) (29.6 %)b (25.2 %)
(7.9 %) (47.7 %) (32.3 %) (58.1 %) (21.7 %)
(75.4 %) (6.9 %) (70.3 %) (10.1 %) (6.2 %) (53.8 %) (21.5 %) (42.3 %) (40.0 %) (5.8 %) (67.6 %)
(42.6 %) (67.1 %) (13.3 %) (5.9 %) (50.4 %) (4.4 %) (19.2 %) (63.3 %) (18.0 %) (63.2 %) (18.1 %)
(9.7 %) (31.2 %) (39.6 %) (22.4 %) (58.0 %) (37.6 %)
2 15 11 17 9
17 6 22 5 0 16 8 10 13 1 23
10 22 6 0 14 4 9 17 9 20 7
1 7 20 13 15 18
1
(1.4 %) (11.5 %) (8.5 %) (13.2 %) (7.0 %)
(13.1 %) (4.6 %)i (15.9 %) (3.7 %) (0.0 %) (12.3 %) (6.2 %) (7.7 %) (10.0 %) (0.7 %) (16.6 %)
(7.1 %) (15.4 %) (4.2 %) (0.0 %) (10.4 %) (3.0 %) (6.7 %) (12.2 %) (6.5 %) (13.9 %) (4.8 %)
(0.7 %) (4.9 %) (13.9 %) (9.1 %) (10.5 %) (12.7 %)
(68.1 %) (3.4 %) (67.5 %) (4.0 %) (3.4 %) (53.0 %) (15.4 %) (44.4 %) (29.1 %) (7.2 %) (60.8 %)
(50.4 %) (61.1 %) (11.5 %) (5.8 %) (57.0 %) (0.8 %) (9.1 %) (68.8 %) (3.2 %) (60.6 %) (11.4 %)
(9.8 %) (33.8 %) (28.6 %) (8.5 %) (63.1 %) (21.7 %)
3 (2.4 %) 58 (45.6 %) 33 (26.0 %)
79 4 85 5 4 62 18 52 34 9 76
65 80 15 7 69 1 11 86 4 80 15
13 45 38 11 82 28
0
Fascin N (%)
9 35 21 43 13
52 3 54 4 6 32 15 30 24 2 48
32 53 9 6 38 2 10 51 8 49 12
(4.6 %)a (22.1 %) (5.3 %) (0.0 %) (6.6 %) (5.0 %) (15.7 %)a (10.4 %) (17.6 %)a (17.4 %) (10.6 %)g (20.7 %) (7.8 %)a (19.8 %) (8.7 %)a (0.8 %) (17.1 %) (10.3 %) (6.8 %) (19.7 %)a (0.8 %) (21.6 %)
6 25 30 13 47 28
(6.77 %) (28.00 %) (16.80 %) (34.96 %) (10.57 %)
(41.60 %) (2.40 %) (40.91 %) (3.03 %) (4.80 %) (25.60 %) (12.00 %) (24.19 %) (19.35 %) (1.50 %) (36.09 %)
(23.88 %) (38.97 %) (6.62 %) (4.7 %) (29.7 %) (1.6 %) (7.8 %) (38.06 %) (5.97 %) (35.77 %) (8.76 %)
(4.4 %) (18.25 %) (21.90 %) (9.49 %) (34.31 %) (20.90 %)
CD44-/ CD24(1.5 %) (3.0 %) (23.3 %)a (20.0 %) (8.4 %)a (23.36 %)
9 (7.2 %)a 18 (14.2 %) 18 (14.2 %)
24 9 25 11 1 20 12 8 23 1 27
6 29 7 0 8 6 19 13 22 23 14
2 4 31 26 11 30
1
1 6 2 6 2
8 0 8 1 0 6 2 3 5 0 8
4 6 3 0 7 1 1 7 2 6 2
1 3 5 2 7 5
(0.75 (4.80 (1.60 (4.88 (1.63
(6.40 (0.00 (6.06 (0.76 (0.00 (4.80 (1.60 (2.42 (4.03 (0.00 (6.02
%) %) %) %) %)
%) %) %) %) %) %) %) %) %) %) %)
(2.99 %) (4.41 %) (2.21 %) (0.0 %) (5.5 %) (0.8 %) (0.8 %) (5.22 %) (1.49 %) (4.38 %) (1.46 %)
(0.7 %) (2.19 %) (3.65 %) (1.46 %) (5.11 %) (3.73 %)
CD44-/ CD24?
CD44/CD24 phenotype N (%)
2 24 21 29 14
42 6 38 10 1 35 13 22 26 5 42
25 38 10 2 27 4 14 34 16 38 13
8 18 24 18 33 23
(1.50 %) (19.20 %) (16.80 %) (23.58 %) (11.38 %)
(33.60 %) (4.80 %) (28.79 %) (7.58 %) (0.80 %) (28.00 %) (10.40 %) (17.74 %) (20.97 %) (3.76 %) (31.58 %)
(18.66 %) (27.94 %) (7.35 %) (1.6 %) (21.1 %) (3.1 %) (10.9 %) (25.37 %) (11.94 %) (27.74 %) (9.49 %)
(5.84 %) (13.14 %) (17.52 %) (13.14 %) (24.09 %) (17.16 %)
CD44 ?/ CD24-
1 7 9 9 7
9 5 13 4 0 10 5 6 8 1 14
5 14 3 0 6 3 7 9 7 13 4
0 2 15 10 7 12
(0.75 (5.60 (7.20 (7.32 (5.69
%) %) %) %) %)
(7.20 %) (4.00 %)j (9.85 %) (3.03 %) (0.00 %) (8.00 %) (4.00 %) (4.84 %) (6.45 %) (0.75 %) (10.53 %)
(3.73 %) (10.29 %) (2.21 %) (0.0 %) (4.6 %) (2.3 %) (5.5 %) (6.72 %) (5.22 %)f (9.49 %) (2.92 %)
(0.00 %) (1.46 %) (10.95 %) (7.30 %) (5.11 %)c (8.96 %)
CD44 ?/ CD24?
In case of significant difference values were bolded Protein expression : 0—lack of expression. 1—expression observed, breast cancer subtype: (1) luminal A (LA): ER? and PR? and HER2- and MIB-1 LI B 14 %, (2) luminal B (LB): ER? and PR? and HER2and MIB-1 LI [ 14 % or ER? and PR? and HER2?, (3) HER2 overexpressing (HER2?): ER- and PR- and HER2?, (4) triple negative phenotype (TNP): ER- and PR- and HER2Lymphocytic infiltration: 0—lack or weak, TS—in tumour stroma, TC—strong among tumour cells a p = 0.000, b p = 0.013, c p = 0.026, d p = 0.044, e p = 0.005, f p = 0.036, g p = 0.006, h p = 0.022, i p = 0.016, j p = 0.010, k p = 0.020, l p = 0.039
Fascin
Laminin-5
Lymphocytic infiltration
P-cadherin
Ep-CAM
SMA
EGFR
CK8/18
CK5/6
Breast cancer subtype (St. Gallen 2011)
HER2
PR
ER
Grade
0
0
1
CD24 N (%)
CD44 N (%)
Table 3 Relations between distribution of different protein expression in CD24, CD44, fascin-positive and -negative tumours
40 J Mol Hist (2014) 45:35–45
J Mol Hist (2014) 45:35–45
expression of fascin, laminin-5, basal markers, lymphocytic infiltration) statistically significantly influenced survival of breast cancer patients (Table 4). Cox multivariate analysis confirmed that pN1 and pN2 as well as CD44?/CD24- immunophenotype are independent positive prognostic factors influencing disease-free survival of breast cancer patients (Table 5). Similar results were obtained when overall survival was analysed (CD44/ CD24 at significance border) (Table 5). We were not able to assess prognostic significance of CD44/CD24/CK5/6/ER coexpression with Cox analysis, as there was no complete observation (progression) in low risk group. In the abovedescribed situation the Cox model does not work.
Discussion CD24 is a small protein which contains 27 amino acids. It is heavily glycosylated and different glycosylation patterns may significantly influence its biological roles (Lim 2005; Lim and Oh 2005; Kristiansen et al. 2004). As CD24 is the ligand for P-selectin, tumour cells expressing this marker could more easily attach to platelets and form small tumour thrombi. Hence, it is postulated that CD24 might play an important role in the process of metastases formation (Lim 2005; Kristiansen et al. 2004). Expression of this protein was studied in many tumours such as: lung, prostate, stomach, ovarian, colon, and renal cancers (Lim and Oh 2005; Kristiansen et al. 2004). In our study we observed 28 (19.2 %) cases with CD24 and 70 (48.6 %) cases with CD44 expression. Our results are consistent with other authors’ results, who reported CD24 expression in 11.4–42 % of cases (Horiguchi et al. 2010; Ricardo et al. 2011; Surowiak et al. 2006; Park et al. 2010; Wang et al. 2011) and CD44 in 36–59 % of cases (Horiguchi et al. 2010; Ricardo et al. 2011; Park et al. 2010; Wang et al. 2011). Our results confirmed the lack of correlation between CD24 positivity and grade, breast cancer subtype, steroid receptor or basal markers status (Ricardo et al. 2011). In other studies correlation with grade (Athanassiadou et al. 2009; Wang et al. 2011) or tumour size (Athanassiadou et al. 2009; Horiguchi et al. 2010) was found. On the other hand, we have noted CD44 immunonegativity more frequently in LA and LB subtype, ER positive tumours as well as in tumours lacking CK5/6, SMA, P-cadherin expression. Similar results were obtained by other researchers, who reported correlation of CD44 expression with breast cancer subtype (based on ER/PR/HER2 status), CK5 and P-cadherin expression (Ricardo et al. 2011), while others—its relation with grade (Wang et al. 2011) or tumour size (Horiguchi et al. 2010). CD44 is a transmembrane protein that acts as a receptor for hyaluronic acid promoting cell
41
migration (Jaggupilli and Elkord 2012). This marker is widely expressed by cancer cells and is associated with proteins involved in extracellular matrix remodelling, cell adhesion, proliferation, and migration (Jaggupilli and Elkord 2012). Hence, the relation between CD44 negativity and parameters associated with less aggressive breast cancer behavior (LA and LB subtypes, ER positivity, CK5/6, SMA, and P-cadherin negativity) is not surprising. On the basis of separately estimated CD24 or CD44 expression we could not indicate a subgroup of patients with statistically significantly better survival rate. However, longer survival (statistically insignificant) was observed in patients having CD24 immunonegative or CD44 immunopositive tumours. Other researchers reported similar results. A relation between higher survival rate and CD24 negativity was found for: breast carcinomas (Athanassiadou et al. 2009; Horiguchi et al. 2010; Kim et al. 2011; Surowiak et al. 2006), gliomas (Deng et al. 2012) and squamous cell carcinoma of the uterine cervix (Kwon et al. 2007). On the other hand, CD44 expression was related to lower progression risk (Horiguchi et al. 2010; Kim et al. 2011). On the basis of combined CD44/CD24 expression we were able to demonstrate significantly lower progression risk in patients with tumours expressing CD44 and without CD24 expression (CD44?/CD24-) than with other immunophenotypes (CD44-/CD24-, CD44?/CD24?, CD44-/CD24?). Similar results were obtained by Kim et al. (2011). We assessed CD44?/CD24- phenotype on the basis of single immunohistochemical staining for each protein. However, some authors performed double staining for CD24 and CD44 simultaneously on the same slide (Abraham et al. 2005; Ricardo et al. 2011; Sung et al. 2010; Tanei et al. 2009). Applying this staining procedure some authors noted better survival of patients with cancers characterized by \10 % of CD44 positive cells that simultaneously were CD24 negative (Ricardo et al. 2011). Other researchers did not support the above-mentioned findings (Abraham et al. 2005; Sung et al. 2010; Tanei et al. 2009). We did not investigate the impact of CD24 and CD44 on survival in chemotherapy- naı¨ve patients group. Therefore, it is difficult to estimate actual role of these proteins in tumour progression. However, the relation between CD44 immunonegativity and favourable prognostic parameters (luminal A and B subtypes, ER positivity, and negativity for CK5/6, EGFR, SMA, P-cadherin) suggest that CD44 expression might be related to more aggressive breast cancer phenotype. In our study, patients with tumours presenting CD44?/CD24- phenotype treated with chemotherapy based on anthracyclines and taxanes survived longer. It may be surprising that phenotype suggested as characteristic for breast cancer stem cells, was related to longer patients survival (however, it should be stressed that
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42
J Mol Hist (2014) 45:35–45
Table 4 Patients 10-year cumulative disease-free survival (DFS) and overall survival (OS) according to analyzed parameters
Chemotherapy
pT
pN Grade
Breast cancer subtype (St. Gallen 2011)
Ep-CAM
Basal markers CD24 CD44 CD44/CD24
CD44/CD24 Fascin Laminin 5 Lymphocytic infiltration
Anthracyclines
N progressed/ without progression
p value
10-yr DFS (%)
N died/ alive
p value
7/22
0.521
75
4/25
0.714
10-yr OS (%) 85
TAC
14/51
77
11/54
82
Taxanes
3/5
60
2/6
75
1
2/13
87
2/13
2
20/63
74
14/69
81
3
2/2
50
1/3
75
1 2
10/53 6/16
83 66
7/56 3/19
3
8/9
53
7/10
1
1/12
90
0/13
2
9/24
71
6/27
3
13/41
75
10/44
LA
1/4
83
1/4
LB
14/39
72
11/42
77
HER2?
2/5
71
2/5
71
TNP
4/20
83
3/21
0
2/3
60
1/4
1
9/37
79
7/39
2
9/24
72
8/25
0
8/24
73
5/27
1
12/38
75
11/39
0 1
18/60 6/10
0.187
75 63
12/66 5/11
0.120
0
17/37
0.087
66
12/42
0.164
1
6/33
85
4/35
CD44-/CD24-
15/31
CD44-/CD24?
2/4
0.216
0.010
0.206
0.935
0.376
0.980
0.032
65
10/36
67
2/4
0.767
87
0.007
87 85
0.224
100
55 79 80 0.830
80
86 0.431
80 83 74
0.980
82 76 83 67 76 89
0.072
76 67
CD44?/CD24-
2/27
93
1/28
96
CD44?/CD24?
4/5
56
3/6
65
CD44?/CD24-
2/27
Other
21/40
0
14/41
1
8/17
0
16/40
1
7/21
0.005 0.436 0.798 0.490
93
1/28
64
15/46
73
10/45
66
6/19
69
9/47
74
7/21
0.016
96 73
0.417
81 72
0.309
82 74
0
1/6
83
1/6
TS
20/59
74
14/65
0.702
81
67
TC
1/8
88
1/8
88
Protein expression: 0—lack of expression, 1—expression observed Breast cancer subtype: (1) luminal A (LA): ER? and PR? and HER2- and MIB-1 LI B 14 %, (2) luminal B (LB): ER? and PR? and HER2and MIB-1 LI [ 14 % or ER? and PR? and HER2?, (3) HER2 overexpressing (HER2?): ER- and PR- and HER2?, (4) triple negative phenotype (TNP): ER- and PR- and HER2Lymphocytic infiltration: 0—lack or weak, TS—in tumour stroma, TC—strong among tumour cells DFS disease-free survival, OS overall survival, TAC taxanes ? anthracyclines
we did not assess CD44/CD24 co-localization but expression of each marker: CD44 and CD2, individually). The explanation of this phenomenon might be higher sensitivity
123
of tumours expressing markers suggested as indicators of more aggressive breast cancer phenotypes, to anthracyclines and taxanes.
J Mol Hist (2014) 45:35–45
43
Fig. 2 Disease-free survival of patients with invasive ductal breast cancer stratified by: a CD44/CD24 phenotype, b CD44/CD24/CK5/6/ER phenotype Table 5 Cox proportional hazards for predictor of disease-free and overall survival Variable
Value
RR
95 % CI
p
1.6–8.7
0.003
0.0–0.7
0.023
1.8–13.1
0.001
0.0–1.1
0.060
Disease-free survival pN CD44/CD24 phenotype
pN1 ? pN2
1
pN3
3.7
CD44?/CD24-
1
CD44?/CD24?, CD44-/CD24?, CD44-CD24-
5.8
Overall survival pN CD44/CD24 phenotype
pN1 ? pN2
1
pN3
4.9
CD44?/CD24-
1
CD44?/CD24?, CD44-/CD24?, CD44-CD24-
7
This hypothesis of higher sensitivity of CD44?/CD24carcinomas to anthracyclines and taxanes might be confirmed by the fact that in our study DFS and OS for TNP subtype (the most aggressive and with worst prognosis) was comparable or even better than for LA subgroup. Similar results were reported by Rouzier et al. (2005) who found basal-like and HER2? subtypes more sensitive to paclitaxel- and doxorubicin-containing preoperative chemotherapy schedules. Moreover, some studies showed (Hugh et al. 2009; Fountzilas et al. 2012) that treatment with chemotherapy including taxanes (as compared to schedules without microtubule-targeting agents) improved survival of HER2? and TNP (only trend) patients. The above-mentioned effect was not noted in patients with luminal A tumours. Both taxanes and anthracyclines act mostly in proliferating cells, disrupting microtubule function and inhibiting DNA synthesis, respectively. Hence, these agents might potentially be more effective in basallike carcinomas that are characterized by more intense proliferation. The effectiveness of anthracyclines and taxanes might result from higher sensitivity of dynamically proliferating, poorly differentiated TNP or HER2? carcinomas to agents targeting cell cycle machinery (at the same
time, slowly proliferating luminal A tumours might be less sensitive). This sensitivity is not in conflict with lower survival rate observed in chemotherapy-naı¨ve, node-negative patients with the above-mentioned breast cancer subtypes (Niemiec et al. 2013). These results are in concordance with St. Gallen 2011 recommendations. The Panel agreed that luminal A subtype is less responsive to chemotherapy, however, anthracyclines and taxanes should be administered to patients with luminal B, HER2? , and TNP cancers (Goldhirsch et al. 2011). The authors believe that similar mechanism may relate to CD44?/CD24- phenotype: higher sensitivity of this phenotype to anthracyclines and taxanes. We did not find relation between CD44/CD24 and breast cancer subtypes and, hence, we suspect that CD44 ?/CD24- might be an independent indicator of chemotherapy resistance. Data concerning the use of CD44 and CD24 and their predictive value still remain unclear. These controversies could be caused by differences in clinical/histological characteristics of studied patients, different treatment schedules and methods applied for immunohistochemical stainings evaluation. Therefore, there is a need for further studies assessing prognostic significance of additional stem
123
44
cell markers. One of the promising candidates is aldehyde dehydrogenase (ALDH1) (Ricardo et al. 2011; Tanei et al. 2009). In our study, tumours without fascin expression more frequently were: (1) ER/PR positive, (2) G1 or G2, (3) LA or LB, (4) negative for basal markers (CK5/6, CK8/ 18, EGFR, SMA, P-cadherin), and (5) with lymphocytic infiltration in tumour stroma. Other authors, similarly to us, found a relation between fascin expression and basallike immunophenotype (Rodrı´guez-Pinilla et al. 2006) and ER negativity (Yoder et al. 2005; Grothey et al. 2000). This suggests that fascin expression is related to more aggressive tumour phenotype, what—on the other hand— confirms findings suggesting fascin to be a modulator of breast cancer cells motility and invasiveness [Al-Alwan et al. 2011; Yoder et al. 2005]. In univariate analysis, the lack of fascin expression was related to longer survival (DFS and OS), although this relation did not reach statistical significance (perhaps due to small group or short follow-up). The same tendency was observed by Yoder et al. (2005), who noted better survival (OS and DFS) in patients with fascin-immunonegative tumours. However, other authors failed to show any association between fascin status and survival (Rodrı´guez-Pinilla et al. 2006). Our findings might indicate that this protein is not a marker of sensitivity to chemotherapy schedules containing taxanes and anthracyclines.
Conclusions Tumours with immunophenotypes: CD44?/CD24- and CD44?/CD24-/CK5/6?/ER- might be more sensitive to chemotherapy based on taxanes and/or anthracyclines. Acknowledgments The study was supported by the Polish Ministry of Science and Higher Education; grant number NN401 096137.
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