Breast Cancer Res Treat (2015) 151:541–553 DOI 10.1007/s10549-015-3430-3
PRECLINICAL STUDY
Loss of PTEN expression is associated with aggressive behavior and poor prognosis in Middle Eastern triple-negative breast cancer Shaham Beg1 • Abdul K. Siraj1 • Sarita Prabhakaran1 • Zeenath Jehan1 • Dahish Ajarim3 • Fouad Al-Dayel2 • Asma Tulbah2 • Khawla S. Al-Kuraya1,4
Received: 2 February 2015 / Accepted: 12 May 2015 / Published online: 16 May 2015 Ó Springer Science+Business Media New York 2015
Abstract PTEN is a tumor suppressor that negatively regulates the PI3 K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In this study, we analyzed PTEN alteration in a tissue microarray format containing more than 1000 primary breast cancers with clinical follow-up data. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry and fluorescence in situ hybridization. Loss of PTEN immunostaining was observed in 77 % of the cases. Shaham Beg and Abdul K Siraj have contributed equally to this work.
PTEN loss was significantly associated with large tumor size (p = 0.0030), high grade (p = 0.0281), tumor recurrence (p = 0.0333), and triple-negative breast cancers (p = 0.0086). PTEN loss in triple-negative breast cancers was significantly associated with rapid tumor cell proliferation (p = 0.0396) and poor prognosis (p = 0.0408). PTEN deletion was found only in 60 cases (6.4 %). Loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore, PTEN signaling pathway might be used as potential therapeutic target in triple-negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer. Keywords
Breast cancer PTEN Triple negative
Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3430-3) contains supplementary material, which is available to authorized users. & Khawla S. Al-Kuraya
[email protected] Shaham Beg
[email protected]
Asma Tulbah
[email protected] 1
Abdul K. Siraj
[email protected]
Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, MBC#98-16, P.O. Box 3354, Riyadh 11211, Saudi Arabia
2
Sarita Prabhakaran
[email protected]
Department of Pathology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
3
Zeenath Jehan
[email protected]
Oncology Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
4
Al-Faisal University, Riyadh, Saudi Arabia
Dahish Ajarim
[email protected] Fouad Al-Dayel
[email protected]
123
542
Abbreviations TNBC Triple-negative breast cancer BLBC Basal-like breast cancer ER Estrogen receptor PR Progesterone receptor HER-2 Human epidermal growth factor receptor 2 IHC Immunohistochemistry FISH Fluorescent in situ hybridization
Introduction Breast cancer is the most common cancer affecting female population worldwide and is the major cause of cancer mortality among females [1]. In Saudi women, breast cancer is ranked first among all cancers [2]. Breast cancers originating from this ethnic population are found to have an advanced stage, advanced local spread, and tend to affect a younger age group as compared to the western population [3, 4]. Breast cancer is very heterogeneous group of diseases whereby many subtypes have been identified based on their morphological features, response to treatment, and clinical outcome [5]. Among the various phenotypes of breast cancers, triple-negative breast cancer (TNBC), characterized by lack of hormone, and Her-2 receptors, is one of the most aggressive breast cancer subtype with high rate of tumor recurrence and the poorest clinical outcomes [6]. TNBC are known to be associated with younger age, late stage at presentation, higher incidences of metastasis, and recurrence [7]. Patients with TNBC can develop brain metastases early in the course of their illness [8]. A major cause of metastasis and recurrence in TNBC may be due to lack of molecular targets for targeted therapy thereby limiting the role of hormonal and biological therapy. Therefore, identification of definitive molecular and prognostic marker is very important to select treatment for this aggressive subtype. PTEN is a tumor suppressor gene on chromosome 10q23 and is one of the key regulators of the PI3 K/Akt pathway activation. PTEN inhibits AKT phosphorylation thereby regulating the PI3 K/Akt pathway [9]. PTEN loss is a frequent event in breast cancer with TNBC showing the highest loss than other breast cancer subgroups, and is significantly associated with younger age at onset of breast cancer and late stage [10, 11]. Loss of PTEN in breast cancers has been associated with poor response to treatment and worse disease outcome through substantial increase in Akt signaling [12]. PTEN loss and high Ki-67 are associated with poor disease-free survival and are independent predictive factors of disease recurrence in breast
123
Breast Cancer Res Treat (2015) 151:541–553
cancers [13, 14]. A recent study has suggested that PTEN loss assessed using IHC might be surrogate markers that can be used to predict outcomes to taxane plus trastuzumab treatment in patients with HER2-overexpressing metastatic breast cancers [15]. Moreover, PTEN analysis by IHC has also been proved to be a more cost-effective and quicker technique than molecular genetic techniques for detecting PTEN status [16]. In our attempt to shed light on classification of breast cancer subtypes in Saudi Arabian population and to find better molecular target that affects therapy or have possible prognostic role, we studied the landscape of PTEN alterations in breast cancers in this ethnic group. The current study was conducted, first to analyze the molecular subtype of breast cancers and finally to investigated the incidence of PTEN protein expression loss and PTEN deletions in 1009 Middle Eastern breast cancers using IHC and FISH. In particular, we have studied the role of PTEN alterations in the Triple-Negative group and this study is among very few studies that present such comprehensive analysis on clinical association between TNBC and PTEN loss along with survival data.
Materials and methods Patient samples and data collection A total of 1009 patients diagnosed with primary breast cancer between 1990 and 2012 were selected from the files of King Faisal Specialist Hospital & Research Centre, Riyadh. Detailed clinico-pathological parameters were noted from case records and integrated clinical information system (ICIS) as described in Table 1. Histological slides were retrieved and reviewed along with the corresponding paraffin block. Patient follow-up was done using clinical data chart and ICIS. Overall survival (OS) was calculated from date of surgery till death or date of last contact with patient if known to be alive. Waiver of consent was obtained from Research Ethics Committee (REC) of hospital for the study. This is a retrospective study performed on archival formalin fixed paraffin-embedded (FFPE) breast cancer samples collected from Department of Pathology and approved by the Basic Research Committee (BRC) and Research Ethics Committee of King Faisal Specialist Hospital and Research Centre under Research Advisory Committee number (RAC # 2040 004). Tissue microarray construction For each tumor, representative areas were selected and mapped, and tissue microarray was constructed. The tissue microarrayer (Semiautomated Arrayer, CM1 Mirlacher,
Breast Cancer Res Treat (2015) 151:541–553
543
Table 1 Correlation of hormone receptor-subtypes with clinico-pathological parameters in breast cancer All cases n (%) Total number of cases
HR?/HER2n (%)
HR?/HER2? n (%)
TNBC n (%)
HR-/HER2?
p value
n (%)
1004 (100)
478 (47.6)
240 (23.9)
149 (14.8)
137 (13.6)
\50
683 (68.0)
301 (44.1)
163 (23.8)
114 (16.7)
105 (15.4)
[50
321 (32.0)
177 (55.1)
77 (24.0)
35 (10.9)
32 (10.0)
Age* 0.0013
Tumor size* B2 cm
219 (22.3)
106 (48.4)
60 (27.4)
23 (10.5)
30 (13.7)
[2 cm
762 (77.7)
362 (47.5)
173 (22.7)
121 (15.9)
106 (13.9)
Negative
311 (33.0)
150 (48.2)
68 (21.9)
59 (19.0)
34 (10.9)
Positive Metastasis*
630 (67.0)
297 (47.1)
160 (25.4)
77 (12.2)
96 (15.2)
M0
810 (90.0)
400 (49.4)
193 (23.8)
115 (14.2)
102 (12.6)
M1
90 (10.0)
34 (37.8)
19 (21.1)
21 (23.3)
16 (17.8)
0.1591
Lymph nodes* 0.0159
0.0439
Tumor stage* I
78 (8.9)
38 (48.7)
24 (30.8)
11 (14.1)
5 (6.4)
II
385 (44.1)
207 (53.8)
79 (205)
55 (14.3)
44 (11.4)
III
319 (36.6)
143 (44.8)
84 (26.3)
41 (12.8)
51 (16.0)
IV
90 (10.3)
34 (37.8)
19 (21.1)
21 (23.3)
16 (17.8)
0.0095
Histological grade * 81 (8.2)
66 (81.5)
13 (16.0)
1 (1.2)
1 (1.2)
Moderately differentiated
Well differentiated
507 (51.1)
265 (52.3)
148 (29.2)
40 (7.9)
54 (10.6)
Poorly differentiated
405 (40.8)
138 (34.1)
78 (19.3)
107 (26.4)
82 (20.2)
914 (93.7)
410 (44.9)
229 (25.1)
141 (15.4)
134 (14.7)
\0.0001
Histology* Infiltrating ductal carcinoma Infiltrating lobular
46 (4.7)
37 (80.4)
6 (13.0)
15 (1.5)
12 (80.0)
2 (13.3)
Yes
368 (41.3)
172 (46.7)
94 (25.5)
45 (12.2)
57 (15.5)
No
522 (58.7)
261 (50.0)
116 (22.2)
86 (16.5)
59 (11.3)
Yes
273 (33.0)
124 (45.4)
70 (25.6)
41 (15.0)
38 (13.9)
No
554 (67.0)
266 (48.0)
140 (25.3)
72 (13.0)
76 (13.7)
Yes
259 (29.3)
114 (44.0)
63 (24.3)
39 (15.1)
43 (16.6)
No
626 (70.7)
315 (50.3)
147 (23.5)
93 (14.9)
71 (11.3)
Mucinous Ca LVI*
2 (4.3) 0
\0.0001
1 (2.2) 1 (6.7) 0.0708
ETE 0.8456
Recurrence 0.1486
Ki-67* High
624 (63.5)
232 (37.2)
143 (22.9)
137 (22.0)
112 (18.0)
Low
358 (36.5)
235 (65.6)
87 (24.3)
12 (3.3)
24 (6.7)
749 (77.8)
380 (50.7)
182 (24.3)
95 (12.7)
92 (12.3)
214 (22.2)
76 (35.5)
46 (21.5)
52 (24.3)
40 (18.7)
\0.0001
p-AKT (ser-473) Low High PTEN IHC Normal
217 (22.7)
88 (40.6)
72 (33.2)
21 (9.7)
36 (16.6)
Loss
737 (77.3)
363 (49.2)
154 (20.9)
123 (16.7)
97 (13.2)
60 (6.5)
15 (25.0)
19 (31.7)
17 (28.3)
9 (15.0)
\0.0001
0.0002
PTEN FISH Deleted
0.0012
123
544
Breast Cancer Res Treat (2015) 151:541–553
Table 1 continued All cases Normal
869 (93.5)
HR?/HER2-
HR?/HER2?
TNBC
HR-/HER2?
426 (49.0)
198 (22.8)
123 (14.1)
122 (14.1)
87.2
77.8
71.7
67.6
p value
Survival OS 5 years
\0.0001
*Data were not available (NA) for some cases: tumor size (NA = 23), lymph nodes (NA = 63), metastasis (NA = 104), stage (NA = 132), grade (NA = 11), histology (NA = 29), LVI (NA = 114), ETE (NA = 177), recurrence (NA = 119), Ki-67 (NA = 22), p-AKT (ser-473) (NA = 41), PTEN IHC (NA = 50), & PTEN FISH (NA = 75)
Neuenburg, Germany) was used to prepare a tissue microarray block as described previously [17]. Briefly, 0.6mm-diameter punches were obtained from the donor blocks and the punched-out tissue cores from the donor block were inserted in the recipient block. The array blocks were incubated at 45 °C for 10 min, cut at room temperature with a standard microtome (Thermo Shandon, Cheshire, United Kingdom) and slides were prepared using tape sectioning system (Instrumedics, Inc., St. Louis, MO). TNBC was defined as ER, PR negative (cut off of nuclear positivity C1 %) and Her-2 negative [18]. Her 2-overexpression was assessed according to ASCO/CAP guidelines [19].
The FISH analyses for PTEN were performed independently and without knowledge of the immunohistochemistry result. The PTEN/CEP17 ratios were calculated as stated in the manufacturer’s guidelines, A cell with two signals of green (centromere 10), and two signals of orange (PTEN) was considered as normal or a PTEN/CEP17 ratio of 1 was considered normal, a tumor cell with two green (centromere 10), and one orange signal (PTEN) with a ratio of 0.5 was considered hemizygous deletion, a tumor cell with two green and total absence of orange signal (PTEN) and a ratio of 0 was considered homozygous deletion.
PTEN & Her 2 FISH
Sections from TMA blocks were subjected to immunohistochemical staining using routine protocol. Sections of 5 lm from the array blocks were cut, slides were deparaffinized in xylene and rehydrated in pure ethanol. Endogenous peroxidase activity was blocked using 3 % hydrogen peroxidase in distil water for 10 min. Antigen retrieval was done by placing the slides in either citrate buffer (pH 6.0) or EDTA (pH 9.0) and placing them in pressure cooker for 5 min at 120 °C. Sections were then subjected to primary antibody; bound antibody was detected with biotinylated link antibody (Dako, Carpinteria, CA) and horseradish peroxidase–labeled streptavidin (Dako). Color was developed in 3, 30 -diaminobenzidine (DAB) with H2O2 as substrate (Dako). Antibody clone, manufacturer and dilution used for each antibody are given in supplementary Table 1. PTEN immunoscoring was done using H-score. Both antibody intensity score of 0, 1?, 2?, 3? (no stain, weak, moderate, and strong staining, respectively) and percentage of positive staining tumor cells was calculated. H-score = 19 (% light staining) ? 29 (% moderate staining) ? 39 (% strong staining). This H-score ranges from 0 to 300. X-tile plots were constructed and cut-off point determined as done previously [20]. Both nuclear and cytoplasmic staining was taken into consideration for scoring. Staining in stromal fibroblast served as positive internal control (Fig. 1). Cases with H-score\90 were taken as loss
FISH for Her2 gene was performed utilizing the PathVysionVysis LSI HER-2/neu Kit from Vysis (Abott Molecular, IL, USA) according to the manufacturers recommendations. The slides were pre-processed with a paraffin pre-treatment reagent kit as per manufacturer recommendation. Slides were screened using the BX51 Olympus fluorescence microscope (Olympus, Richardson, TX) and a SenSysW CCD camera (Photometrics, Tucson, AZ) was used to capture selected images. Twenty to thirty tumor nuclei per core, with both red and green signals were selected for scoring. The ratio of Her-2 to chromosome 17 centromeric region of [2.2 was considered as amplified. For PTEN dual-color FISH on paraffin-embedded TMA was performed using commercially available DNA probes LSI PTEN/CEP 10; VysisInc. The PTEN locus specific probe located on cytoband10q23 labeled with Spectrum Orange and centromere of chromosome 10 probe region 10p11.1-q11.1 labeled with Spectrum Green (LSI PTEN/ CEP 10; Vysis Inc.). The PTEN genomic probe spans 368 kb and starts 166 kb from 50 end of the gene and extends 98 kb past the 30 end of the gene. Histologic tissue microarray tissue Sects. 5 lm thick were deparaffinized with a series of xylene prior to immersion in 100 % ethanol. FISH was carried according to the manufacturer’s instructions.
123
Immunohistochemistry
Breast Cancer Res Treat (2015) 151:541–553
545
Fig. 1 Immunohistochemical analysis of PTEN, p-AKT, and PTEN FISH deletion in breast cancer patients. TMA spots showing overexpression of a PTEN and c p-AKT. In contrast, different TMA spots showing reduced/absent expression of b PTEN and d pAKT. e FISH image shows normal cells with two orange normal
signals (PTEN) and two green centromere 10 signals. f FISH image shows tumor cells with no orange signal (PTEN) having homozygous deletion and two green centromere 10 signals. 209 magnification on Olympus BX-51 microscope (Olympus America, Center Valley, PA, USA) with inset showing 409 magnifications of the same TMA spot
of PTEN. We chose H-score method as it is considered to be a better IHC scoring method compared to intensity score for PTEN staining [21]. p-AKT was considered positive if
any staining in tumor tissue was seen with intensity score of C2. Basal Cytokeratin (CK 14, 34be12) and EGFR were considered positive for any staining in C1 % of tumor
123
546
Breast Cancer Res Treat (2015) 151:541–553
Fig. 2 Bar diagram showing proportion of PTEN expression loss by IHC in different molecular subtypes of Middle Eastern breast cancer
cells. For Ki 67 nuclear staining was considered as positive. The cutoff for high Ki67 was taken as more than 30 % of tumor nuclei staining in the total tumor area. We defined basal-like breast cancer (BLBC) as tumors showing negativity for ER, PR, & Her-2 and positivity for any one of the basal markers (CK14, EGFR, and 34be12) in subgroups as defined previously [22]. IHC scoring was done by two pathologists (SB & SP), blinded to the clinico-pathological characteristics. Discordant scores were reviewed together to achieve agreement. Statistical analysis The JMP 10.0 (SAS Institute Inc., Cary, NC) software package was used for data analyses. We examined the association of TNBC with clinico-pathological parameters, biomarker expression and also performed survival analysis. Survival curves were generated using Kaplan–Meier method with significance evaluated using the Mantel-Cox log-rank test. Risk ratio was calculated using the Cox proportional hazard model in both univariate and multivariate analyses. Values of p \ 0.05 were considered statistically significant.
Results Characterization of breast cancer One thousand and nine cases diagnosed with primary breast carcinoma at King Faisal Specialist Hospital & Research Center were investigated for the clinico-pathological
123
parameters and molecular profile for the incidence of various molecular subtypes of breast cancers. Majority of the cases were between 30 and 60 years age group and the median age at the time of surgery was 45 years (inter quartile range (IQR), 39.0–54.0 years). The median length of follow-up available for surviving patients was 53.0 months (IQR, 30–77 months). The 5-year OS rate was 80.5 %. Infiltrating duct carcinoma was the most common histological subtype and constituted 93.7 % of total cases followed by lobular carcinoma which was 4.7 %. Majority of tumors were moderately differentiated (51.1 %) and most of the patients were in stage II (44.1 %) at the time of diagnosis followed by stage III (36.6 %), stage IV (10.3 %), and stage I (8.9 %). At the time of diagnosis 67.0 %of the cases showed lymph node metastasis. Breast cancer subtypes In our cohort of breast cancer cases, 71.5 % (720/1006) of cases were hormone receptor (HR) positive (ER? and/or PR?). Her 2? positive tumors constituted 28.6 % (273/ 956) of our cohort. Based on hormone receptors and Her-2 status, the most common subtypes of breast cancer in our cohort were hormone receptors positive and Her2 negative (HR?/Her2-) tumors which constituted 47.6 % (478/ 1004) of our cases. Of the remaining cases, 23.9 % (240/ 1004) were hormone receptors positive & Her-2 positive, 14.8 % (149/1004) were negative for both hormone receptors and Her-2 (TNBC) and 13.6 % (137/1004) of tumors were hormone receptors negative & Her-2 positive. Breast cancer subtypes were found to be significantly associated with age (p = 0.0013), lymph node status
Breast Cancer Res Treat (2015) 151:541–553
547
Table 2 Correlation of PTEN IHC with clinico-pathological parameters in breast cancer Total N Total number of cases
PTEN loss %
957
PTEN expression
p value
N
%
N
%
737
77.0
220
23.0
Age \50
651
68.0
508
78.0
143
22.0
[50
306
32.0
229
74.8
77
25.2
207
22.1
143
69.1
64
30.9
730
77.9
578
79.2
152
20.8
Negative
296
33.0
233
78.7
63
21.3
Positive
600
67.0
465
77.5
135
22.5
0.2756
Tumor size* B2 cm [2 cm Lymph nodes*
0.0030
0.6791
Metastasis* M0
768
89.7
584
76.0
184
24.0
M1
88
10.3
63
71.6
25
28.4
0.3645
Tumor stage* I
72
8.7
50
69.4
22
30.6
II
364
43.9
280
76.9
84
23.1
III
305
36.8
233
76.4
72
23.6
IV
88
10.6
63
71.6
25
28.4
0.4579
Histological grade* Well differentiated
74
7.8
53
71.6
21
28.4
486
51.3
361
74.3
125
25.7
388
40.9
315
81.2
73
18.8
873
93.9
669
76.6
204
23.4
Infiltrating lobular
41
4.4
37
90.2
4
9.8
Mucinous Ca
16
1.7
9
56.3
7
43.7
Yes
352
41.5
260
73.9
92
26.1
No
496
58.5
385
77.6
111
22.4
Yes
263
33.6
204
77.6
59
22.4
No
521
66.4
392
75.2
129
24.8
Yes
243
28.8
196
80.7
47
19.3
No
600
71.2
443
73.8
157
26.2
329
34.4
262
79.6
67
20.4
627
65.6
475
75.8
152
24.2
Negative
402
42.2
320
79.6
82
20.4
Positive
551
57.8
416
75.5
135
24.5
Negative
683
71.4
549
80.4
134
19.6
Positive
273
28.6
188
68.8
85
31.2
Yes
144
15.1
123
85.4
21
14.6
No
808
84.9
613
75.9
195
24.1
Moderately differentiated Poorly differentiated Histology* Infiltrating ductal carcinoma
0.0281
0.0158
LVI* 0.2076
ETE* 0.4696
Recurrence* 0.0333
ER* Negative Positive PR*
0.1724
0.1343
HER-2 IHC* 0.0002
Triple negative* 0.0086
123
548
Breast Cancer Res Treat (2015) 151:541–553
Table 2 continued Total
PTEN loss
PTEN expression
p value
N
%
N
%
N
%
High
603
64.3
469
77.8
134
22.2
Low
334
35.7
256
76.7
78
23.3
Ki-67* 0.6923
p-AKT (ser-473)* Low
728
77.6
560
76.9
168
23.1
High
210
22.4
167
79.5
43
20.5
0.4228
PTEN FISH* Deleted
60
6.7
51
85.0
9
15.0
830
93.3
633
76.3
197
23.7
Yes
129
13.5
110
85.3
19
14.7
No
828
86.5
627
75.7
201
24.3
Normal/gain
0.1049
Basal-like breast cancer 0.0123
Survival OS 5 years
79.7
81.3
0.2644
*Data were not available (NA) for some cases: tumor size (NA = 20), lymph nodes (NA = 61), metastasis (NA = 101), stage (NA = 128), grade (NA = 09), histology (NA = 27), LVI (NA = 109), ETE (NA = 173), recurrence (NA = 114), ER (NA = 01), PR (NA = 04), HER-2 IHC (NA = 01), triple negative (NA = 05), Ki-67 (NA = 20), p-AKT (ser-473) (NA = 19) & PTEN FISH (NA = 67)
(p = 0.0159), distant metastasis (p = 0.0439), tumor stage (p = 0.0095), tumor grade (p \ 0.0001), Ki-67 (p \ 0.0001), p-AKT (p \ 0.0001), PTEN IHC loss (p = 0.0002), and PTEN FISH deletion (p = 0.0012) (Table 1). Basal-like breast tumor comprised 13.3 % (134/ 1009) of our cohort and constituted 89.9 % of TNBC cases. PTEN and breast cancer PTEN loss by IHC was seen in 77 % (737/957) of breast cancer cases (Fig. 2). PTEN loss in breast tumors were significantly associated with clinical parameters such as large tumor size (p = 0.0030), high tumor grade (p = 0.0281), histological subtype (p = 0.0158), and tumor recurrence (p = 0.0333). Significant direct associations of PTEN loss was seen with TNBC (p = 0.0086) and basal-like tumors (p = 0.0123). PTEN loss by IHC was also strongly associated with Her-2 negative tumors (p = 0.0002) (Table 2). PTEN deletion by FISH was seen in only 6.4 % (60/934) of breast cancer cases, all the PTEN deleted cases belonged to infiltrating ductal subtype. Significant direct association of PTEN deletion was seen with TNBC, basal-like tumors and inverse association with ER and PR hormone receptors. An association was also seen between PTEN deletion and p-AKT but this could not reach statistical significance (p = 0.0642).There was no correlation seen between PTEN loss by IHC and PTEN deletion by FISH (Supplementary Table 2).
123
PTEN and triple-negative breast cancer The incidence of PTEN loss by IHC in TNBC was 85.4 % (123/144). PTEN loss was associated with younger age (p = 0.0334) and a trend was seen with tumor recurrence (p = 0.0736). PTEN loss was significantly associated with proliferative marker Ki-67 (p = 0.0396) (Table 3). PTEN deletion by FISH was seen in 12.1 % (17/140) of TNBC cases and no significant association was seen with any clinical and molecular parameter. All 17 PTEN deleted cases showed loss of PTEN protein expression on IHC (p = 0.0191) (Supplementary Table 3). Survival Survival analysis of different breast cancer subgroups showed that patients that were HR negative & Her-2 positive tumors had significantly poor 5-year OS (67.6 %) compared to all the other groups (p \ 0.0001). The OS of this group was even poorer than TNBC. Interestingly PTEN expression loss by IHC and PTEN deletion by FISH were not associated with significant survival difference in our cohort of breast cancer cases and also among different HR/Her-2 subgroups, except for the TNBC group. PTEN expression loss in TNBC was associated with significant poor 5-year OS compared to TNBC tumors with normal PTEN protein expression (p = 0.0408). Also among TNBC tumors, cases showing both expression loss and PTEN deletion showed worse survival outcome compared to cases with normal PTEN
Breast Cancer Res Treat (2015) 151:541–553
549
Table 3 Correlation of PTEN IHC in triple-negative with clinico-pathological parameters in breast cancer Total N Total number of cases
PTEN loss %
144
N
PTEN expression %
N
p value
%
123
85.4
21
14.6
Age \50
110
76.4
98
89.1
12
10.9
[50
34
23.6
25
73.5
9
26.5
0.0334
Tumor size* B2 cm
23
16.4
21
91.3
2
8.7
117
83.6
98
83.8
19
16.2
Negative
59
44.7
51
86.4
8
13.6
Positive
73
55.3
61
83.6
12
16.4
[2 cm Lymph nodes*
0.3267
0.6453
Metastasis* M0
111
84.1
95
85.6
16
14.4
M1
21
15.9
17
81.0
4
19.0
0.5966
Tumor stage* I
11
8.9
10
90.9
1
9.1
II
54
43.6
44
81.5
10
18.5
III
38
30.6
34
89.5
4
10.5
IV
21
16.9
17
81.0
4
19.0
0.6295
Histological grade* Well differentiated Moderately differentiated Poorly differentiated Histology* Infiltrating ductal carcinoma
1
0.7
0
0
1
100.0
38
26.6
34
89.5
4
10.5
104
72.7
88
84.6
16
15.4
136
98.6
116
85.3
20
14.7
Infiltrating lobular
2
1.4
2
100.0
0
0
Mucinous Ca
0
0
0
0
0
0
Yes
43
34.1
38
88.4
5
11.6
No
83
65.9
70
84.3
13
15.7
Yes
40
36.7
37
92.5
3
7.5
No
69
63.3
58
84.1
11
15.9
Yes
38
29.7
35
92.1
3
7.9
No
90
70.3
72
80.0
18
20.0
134
93.1
117
87.3
17
12.7
10
6.9
6
60.0
4
40.0
Low
92
64.3
78
84.8
14
15.2
High
51
35.7
44
86.3
7
13.7
0.1081
0.4269
LVI* 0.5336
ETE* 0.1883
Tumor recurrence* 0.0736
Ki-67* High Low p-AKT (ser-473)*
0.0396
0.8084
PTEN FISH* Deleted Normal/gain
17
12.5
17
100.0
0
0
119
87.5
100
84.0
19
16.0
0.0191
Basal-like breast cancer Yes
129
89.6
110
85.3
19
14.7
No
15
10.4
13
86.7
2
13.3
0.8835
123
550
Breast Cancer Res Treat (2015) 151:541–553
Table 3 continued Total N
PTEN loss %
N
PTEN expression %
N
p value
%
Survival OS 5 years
68.4
93.7
0.0408
* Data were not available (NA) for some cases: Tumor size (NA = 04), Lymph nodes (NA = 12), Metastasis (NA = 12), Stage (NA = 20), Grade (NA = 01), Histology (NA = 06),LVI(NA = 18), ETE (NA = 35), Recurrence (NA = 16), p-AKT(ser-473) (NA = 01), &PTEN FISH (NA = 08)
copy number and normal protein expression (p = 0.0256). There was no survival difference in Her-2? tumors between cases showing PTEN expression loss and normal expression by IHC (p = 0.9404). However, in Her-2 negative tumors PTEN loss showed worse OS compared to tumors expressing PTEN protein but this difference could not reach statistical significance (p = 0.0538). When we analyzed expression of p-AKT, an important protein of the PI3 K/AKT survival pathway, p-AKT activation was found to have significantly poor OS (p = 0.0164) in our cohort of breast cancer cases (Fig. 3).When patients were segregated into groups on the basis of PTEN expression loss and p-AKT activation, patients showing PTEN expression loss and p-AKT activation were found to have significant poor outcome compared to tumors with normal PTEN expression and low p-AKT (p = 0.0316). On multivariate analysis this survival difference was statistically significant (RR = 1.93; 95 % CI 1.05–3.70; p = 0.0341) (data not shown). Significant survival difference was also seen between PTEN loss/pAKT ? and PTEN loss/p-AKT low tumors (p = 0.0403) but this survival difference could not reach statistically significance on multivariate analysis (Fig. 3).
Discussion Using a TMA of more than 1000 clinical samples of breast cancer, we found that a significant proportion of Middle Eastern breast cancer show PTEN expression loss (77 %) and this expression loss is even more common in TNBC (85 %) as detected by IHC. The result of our study is in concordance with earlier published data on PTEN expression loss which range from 22 to 86 % [23, 24]. Such high percentage of PTEN expression loss in our cohort indicates the greater role of tumor suppressor PTEN gene in breast carcinogenesis in this ethnic group. We found a significant direct association between TNBC with PTEN expression loss as well as with PTEN deletion. To the best of our knowledge; this is the second study showing this association in breast cancers. Previously, similar association has been reported on African American TNBC [14]. We
123
also observed that even with stringent scoring criteria, taking cut-off- as any detectable staining, there was no change in the results. PTEN loss was associated with proliferative marker Ki-67 in TNBC cases showing the aggressive behavior of these tumors. In addition, we also found that TNBC with PTEN loss showed poor survival when compared to PTEN expressing TNBC. This finding is in concordance with previously published studies. [11, 25] . In this study 71.4 % of tumors were Her-2 negative and were significantly associated with PTEN loss, similar findings has also been previously reported in breast cancer from other regions [26, 27]. We could not find any prognostic role of PTEN expression in Her-2 positive tumors, on the other hand interestingly enough, we found that PTEN loss showed trend toward poor survival in Her-2 negative tumors (p = 0.0538).Similar to our findings, one of largest study performed on PTEN expression in Her-2 positive breast cancer in 2013 failed to demonstrate any prognostic role of PTEN expression [28]. Phosphorylated-AKT was detected in 22 % of breast cancer cases and this activation was highest in TNBC (35 %) followed by HR-/Her ? tumors (30 %). Interestingly, we could not find any correlation between p-AKT expression and PTEN alteration and this finding was concordant with data shown by other groups [29, 30]. Although PTEN loss alone was not associated with survival in overall breast cancer cases, but patients showing combined PTEN loss and p-AKT had significantly poor OS compared to those with no AKT activation. These findings are similar to the study that was performed on Her-2 positive metastatic breast cancer [31]. Our findings suggest that PTEN loss may not be the only signaling pathway involved in downstream target AKT phosphorylation and involvement of other mechanism is hypothesized. Moreover, in view of our findings importance of activated PI3 K/AKT pathway in PTEN-mediated breast carcinogenesis cannot be undermined. We also found that both PTEN expression loss and gene deletion were associated with basal-like breast carcinomas (BLBC), which are highly malignant poor prognosis breast cancer with overlapping features with TNBC. This PTEN loss in BLBC was observed even more than in any other
Breast Cancer Res Treat (2015) 151:541–553
551
Fig. 3 Kaplan–Meirer survival curves. a In breast cancer patients, there was no significance in survival between PTEN loss and PTEN expressed cases. b Cases with p-AKT activation showed worse survival outcome compared to normal p-AKT. c, d PTEN loss with p-AKT overexpression is associated with significantly poor OS
compared to cases showing normal PTEN and low p-AKT expression and PTEN loss and low p-AKT respectively. e Triple-negative breast cancer showing PTEN loss is associated with poor OS. f Patients with combined PTEN loss and PTEN deletion have worse disease outcome compared to cases with normal PTEN by IHC and FISH
subtypes of breast cancer in this cohort. Significant direct association between PTEN loss and BLBC has also been reported earlier in another study that showed, PI3 K-AKT pathway is activated more in BLBC compared to Her2? tumors and the molecular mechanism involved in such activation is loss of PTEN [32]. It has also been shown that
BLBC with activated PI3 K-AKT pathway are therapeutically targetable by specific inhibitors in preclinical studies [33]. Although we could not find any association between PTEN expression loss and PTEN deletion in this cohort of breast cancer cases; however, this association was present
123
552
in TNBC. Similarly, Razis et al. also could not find any association between PTEN FISH deletion and PTEN expression in their study on 256 breast cancer patients [27]. Moreover, no correlation was detected between PTEN mRNA level and FISH deletion in another study performed by Neto et al. on 59 breast cancer cases [34]. They found only 7 PTEN deleted case on FISH. The disparity in proportion of cases showing protein expression loss and FISH deletion in our study show that deletion is not the only mechanism responsible for low PTEN, but factors such as PTEN gene promoter methylation and mutation may be the dominant mechanism playing a greater role in PTEN-mediated carcinogenesis in this ethnic population. Further genetic studies on PTEN methylation/mutation is an area of research interest in our lab now which will be reflected in future publications.
Conclusion PTEN loss is involved in majority of Middle Eastern breast cancers and is associated with highly aggressive TNBCs. To the best of our knowledge, this is the first and the largest study on Middle Eastern breast cancers interrogating PTEN alteration and its prognostic role in TNBC. Gene deletion accounts for minority of this expression loss and other mechanisms such as promoter methylation and mutation are hypothesized to play a greater role for PTEN-mediated breast carcinogenesis in this ethnic group. PTEN loss is a marker of poor prognosis in TNBC and this PTEN signaling pathway can be therapeutically targeted. Further studies are needed to confirm our findings. Acknowledgments We thank Valorie Balde, Padmanaban Annaiyappanaidu, and Zeeshan Qadri for technical assistance. Conflict of interests All authors declare that they have no conflict interests in this manuscript.
References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A (2015) Global cancer statistics, 2012. CA Cancer J Clin 65(2):87–108. doi:10.3322/caac.21262 2. Bazarbashi S (2010) Cancer Incidence report Saudi Arabia, 35–37 3. Al Tamimi DM, Shawarby MA, Ahmed A, Hassan AK, AlOdaini AA (2010) Protein expression profile and prevalence pattern of the molecular classes of breast cancer–a Saudi population based study. BMC Cancer 10:223. doi:10.1186/1471-2407-10-223 4. Al-Kuraya K, Schraml P, Sheikh S, Amr S, Torhorst J, Tapia C, Novotny H, Spichtin H, Maurer R, Mirlacher M, Simon R, Sauter G (2005) Predominance of high-grade pathway in breast cancer development of Middle East women. Mod Pathol 18(7):891–897. doi:10.1038/modpathol.3800408
123
Breast Cancer Res Treat (2015) 151:541–553 5. Stagg J, Allard B (2013) Immunotherapeutic approaches in triplenegative breast cancer: latest research and clinical prospects. Ther Adv Med Oncol 5(3):169–181. doi:10.1177/1758834012475152 6. Andre F, Zielinski CC (2012) Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents. Ann Oncol 23 Suppl 6:vi46–vi51. doi:10.1093/ annonc/mds195 7. Di Cosimo S, Baselga J (2010) Management of breast cancer with targeted agents: importance of heterogeneity. [corrected]. Nat Rev Clin Oncol 7(3):139–147. doi:10.1038/nrclinonc.2009.234 8. Nam BH, Kim SY, Han HS, Kwon Y, Lee KS, Kim TH, Ro J (2008) Breast cancer subtypes and survival in patients with brain metastases. Breast Cancer Res 10(1):R20. doi:10.1186/bcr1870 9. Hollander MC, Blumenthal GM, Dennis PA (2011) PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 11(4):289–301. doi:10.1038/nrc3037 10. Song CH, Park SY, Eom KY, Kim JH, Kim SW, Kim JS, Kim IA (2010) Potential prognostic value of heat-shock protein 90 in the presence of phosphatidylinositol-3-kinase overexpression or loss of PTEN, in invasive breast cancers. Breast Cancer Res 12(2):R20. doi:10.1186/bcr2557 11. Dean SJ, Perks CM, Holly JM, Bhoo-Pathy N, Looi LM, Mohammed NA, Mun KS, Teo SH, Koobotse MO, Yip CH, Rhodes A (2014) Loss of PTEN expression is associated with IGFBP2 expression, younger age, and late stage in triple-negative breast cancer. Am J Clin Pathol 141(3):323–333. doi:10.1309/ AJCPR11DEAYPTUSL 12. Craig DW, O’Shaughnessy JA, Kiefer JA, Aldrich J, Sinari S, Moses TM, Wong S, Dinh J, Christoforides A, Blum JL, Aitelli CL, Osborne CR, Izatt T, Kurdoglu A, Baker A, Koeman J, Barbacioru C, Sakarya O, De La Vega FM, Siddiqui A, Hoang L, Billings PR, Salhia B, Tolcher AW, Trent JM, Mousses S, Von Hoff D, CarPTEN JD (2013) Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities. Mol Cancer Ther 12(1):104–116. doi:10.1158/1535-7163.MCT-12-0781 13. Capodanno A, Camerini A, Orlandini C, Baldini E, Resta ML, Bevilacqua G, Collecchi P (2009) Dysregulated PI3 K/Akt/PTEN pathway is a marker of a short disease-free survival in nodenegative breast carcinoma. Hum Pathol 40(10):1408–1417. doi:10.1016/j.humpath.2009.02.005 14. Wu Y, Sarkissyan M, Elshimali Y, Vadgama JV (2013) Triple negative breast tumors in African-American and Hispanic/Latina women are high in CD44?, low in CD24?, and have loss of PTEN. PLoS ONE 8(10):e78259. doi:10.1371/journal.pone. 0078259 15. Park YH, Jung HA, Choi MK, Chang W, Choi YL, Do IG, Ahn JS, Im YH (2014) Role of HER3 expression and PTEN loss in patients with HER2-overexpressing metastatic breast cancer (MBC) who received taxane plus trastuzumab treatment. Br J Cancer 110(2):384–391. doi:10.1038/bjc.2013.757 16. Jones N, Bonnet F, Sfar S, Lafitte M, Lafon D, Sierankowski G, Brouste V, Banneau G, Tunon de Lara C, Debled M, MacGrogan G, Longy M, Sevenet N (2013) Comprehensive analysis of PTEN status in breast carcinomas. Int J Cancer 133(2):323–334. doi:10. 1002/ijc.28021 17. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G, Kallioniemi OP (1998) Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med 4(7):844–847 18. Zhang J, Wang Y, Yin Q, Zhang W, Zhang T, Niu Y (2013) An associated classification of triple negative breast cancer: the risk of relapse and the response to chemotherapy. Int J Clin Exp Pathol 6(7):1380–1391 19. Deyarmin B, Kane JL, Valente AL, van Laar R, Gallagher C, Shriver CD, Ellsworth RE (2013) Effect of ASCO/CAP
Breast Cancer Res Treat (2015) 151:541–553
20.
21.
22.
23.
24.
25.
26.
27.
guidelines for determining ER status on molecular subtype. Ann Surg Oncol 20(1):87–93. doi:10.1245/s10434-012-2588-8 Camp RL, Dolled-Filhart M, Rimm DL (2004) X-tile: a new bioinformatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res 10(21):7252–7259. doi:10.1158/1078-0432.CCR-04-0713 Maiques O, Santacana M, Valls J, Pallares J, Mirantes C, Gatius S, Garcia Dios DA, Amant F, Pedersen HC, Dolcet X, MatiasGuiu X (2014) Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues. Hum Pathol 45(3):522–532. doi:10.1016/j.humpath.2013.10.018 Rakha E, Reis-Filho JS (2009) Basal-like breast carcinoma: from expression profiling to routine practice. Arch Pathol Lab Med 133(6):860–868. doi:10.1043/1543-2165-133.6.860 Duman BB, Sahin B, Acikalin A, Ergin M, Zorludemir S (2013) PTEN, Akt, MAPK, p53 and p95 expression to predict trastuzumab resistance in HER2 positive breast cancer. J BUON 18(1):44–50 Yonemori K, Tsuta K, Shimizu C, Hatanaka Y, Hashizume K, Ono M, Kouno T, Ando M, Tamura K, Katsumata N, Hasegawa T, Kinoshita T, Fujiwara Y (2009) Immunohistochemical expression of PTEN and phosphorylated Akt are not correlated with clinical outcome in breast cancer patients treated with trastuzumab-containing neo-adjuvant chemotherapy. Med Oncol 26(3):344–349. doi:10.1007/s12032-008-9127-2 Iqbal J, Thike AA, Cheok PY, Tse GM, Tan PH (2012) Insulin growth factor receptor-1 expression and loss of PTEN protein predict early recurrence in triple-negative breast cancer. Histopathology 61(4):652–659. doi:10.1111/j.1365-2559.2012.04255.x Perez-Tenorio G, Alkhori L, Olsson B, Waltersson MA, Nordenskjold B, Rutqvist LE, Skoog L, Stal O (2007) PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer. Clin Cancer Res 13(12):3577–3584. doi:10.1158/1078-0432.CCR-06-1609 Razis E, Bobos M, Kotoula V, Eleftheraki AG, Kalofonos HP, Pavlakis K, Papakostas P, Aravantinos G, Rigakos G, Efstratiou I, Petraki K, Bafaloukos D, Kostopoulos I, Pectasides D, Kalogeras KT, Skarlos D, Fountzilas G (2011) Evaluation of the association of PIK3CA mutations and PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer. Breast Cancer Res Treat 128(2):447–456. doi:10.1007/s10549-011-1572-5
553 28. Perez EA, Dueck AC, McCullough AE, Chen B, Geiger XJ, Jenkins RB, Lingle WL, Davidson NE, Martino S, Kaufman PA, Kutteh LA, Sledge GW, Harris LN, Gralow JR, Reinholz MM (2013) Impact of PTEN protein expression on benefit from adjuvant trastuzumab in early-stage human epidermal growth factor receptor 2-positive breast cancer in the North Central Cancer Treatment Group N9831 trial. J Clin Oncol 31(17):2115–2122. doi:10.1200/JCO.2012.42.2642 29. Panigrahi AR, Pinder SE, Chan SY, Paish EC, Robertson JF, Ellis IO (2004) The role of PTEN and its signalling pathways, including AKT, in breast cancer; an assessment of relationships with other prognostic factors and with outcome. J Pathol 204(1):93–100. doi:10.1002/path.1611 30. Bose S, Chandran S, Mirocha JM, Bose N (2006) The Akt pathway in human breast cancer: a tissue-array-based analysis. Mod Pathol 19(2):238–245. doi:10.1038/modpathol.3800525 31. Esteva FJ, Guo H, Zhang S, Santa-Maria C, Stone S, Lanchbury JS, Sahin AA, Hortobagyi GN, Yu D (2010) PTEN, PIK3CA, p-AKT, and p-p70S6 K status: association with trastuzumab response and survival in patients with HER2-positive metastatic breast cancer. Am J Pathol 177(4):1647–1656. doi:10.2353/ ajpath.2010.090885 32. Marty B, Maire V, Gravier E, Rigaill G, Vincent-Salomon A, Kappler M, Lebigot I, Djelti F, Tourdes A, Gestraud P, Hupe P, Barillot E, Cruzalegui F, Tucker GC, Stern MH, Thiery JP, Hickman JA, Dubois T (2008) Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells. Breast Cancer Res 10(6):R101. doi:10.1186/bcr2204 33. DeGraffenried LA, Fulcher L, Friedrichs WE, Grunwald V, Ray RB, Hidalgo M (2004) Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/ Akt pathway. Ann Oncol 15(10):1510–1516. doi:10.1093/ annonc/mdh388 34. Neto JC, Ikoma MM, Carvalho KC, Vassallo J, De Brot M, Gobbi H, Soares FA, Rocha RM (2012) MGMT and PTEN as potential prognostic markers in breast cancer. Exp Mol Pathol 92(1):20–26. doi:10.1016/j.yexmp.2011.09.019
123
