Human Pathology (2014) 45, 2437–2446
www.elsevier.com/locate/humpath
Original contribution
Prognostic relevance of estrogen receptor-α Ser167 phosphorylation in stage II-III colon cancer patients☆,☆☆ Iker López-Calderero MD a,b , Amancio Carnero PhD a , Aurora Astudillo MD, PhD c , José Palacios MD, PhD d , Manuel Chaves MD b , Marta Benavent MD a,b , María L. Limón MD b , Rocio Garcia-Carbonero MD, PhD a,b,⁎ a
Instituto de Biomedicina de Sevilla (IBIS), HUVR/CSIC/Universidad de Sevilla, Center affiliated to the Red Temática de Investigación Cooperativa en Cancer (RTICC), Instituto Carlos III, Spanish Ministry of Science and Innovation, Seville, 41013, Spain b Department of Medical Oncology, Hospital Universitario Virgen del Rocío, Center affiliated to the Red Temática de Investigación Cooperativa en Cancer (RTICC), Instituto Carlos III, Spanish Ministry of Science and Innovation, Seville, 41013, Spain c Department of Pathology, Hospital Central de Asturias, Asturias, 33006, Spain d Department of Pathology, Hospital Universitario Virgen del Rocío, Seville, 41013, Spain Received 2 June 2014; revised 22 July 2014; accepted 5 August 2014
Keywords: Colon cancer; Tissue microarray; Estrogen receptor; Immunohistochemistry; Prognosis; Survival; Biomarkers
Summary Preclinical and clinical data suggest a protective role for estrogens on colon cancer (CRC) risk. estrogen receptor (ER) β is the prevalent ER in normal colonic mucosa, whereas its expression is significantly reduced in CRC. An increased ERα/β ratio has been documented in colon carcinomas and is associated with increased proliferation and decreased apoptosis. The aim of our study was to evaluate the expression of activated ERα and its prognostic implications in patients with stage II-III CRC. Phospho-ERαSer167 (pERαSer167) expression was assessed by immunohistochemistry in 218 CRC paraffin-embedded tumor samples. A high pERαSer167 expression was more commonly observed in women, older patients, and patients with high baseline glucose levels. This higher pERαSer167 expression was associated with decreased 5-year disease-free interval (DFI; 66% versus 78%, P = .07) and overall survival (65% versus 73%, P = .46). The negative impact of high pERαSer167 expression on DFI was particularly significant (P b .05) in women (85% versus 60%), young (82% versus 61%), nondiabetic (85% versus 66%), and stage II patients (86% versus 72% and low versus high pERαSer167, respectively). Multivariate analysis confirmed that pERαSer167 score was a significant prognostic factor for both DFI and overall survival, independent of sex, age, glucose levels, tumor stage, bowel obstruction/ perforation, or adjuvant chemotherapy. These findings illustrate the relevance of estrogen pathways in colon cancer biology and may provide novel therapeutic avenues to be explored in this context. © 2014 Elsevier Inc. All rights reserved.
☆
Competing interests: The authors declare no conflicts of interest. Funding/Support: This work was partially supported by a grant from the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III Spanish Cancer Networks RTICC (R12/0036/0008 and R12/0036/0028). R.G.C. is funded by the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (PI10/02164, PI13/02295), Servicio Andaluz de Salud (PI-0259/2007), and Fundacion Mutua Madrileña (P0497/2006). A.C. is funded by the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (FIS PI12/00137), Consejeria de Ciencia e Innovacion (CTS-6844, CM09/00207), and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). ⁎ Corresponding author at: Medical Oncology Department, Hospital Universitario Virgen del Rocio, Laboratorio de Oncologia Traslacional y Nuevas Terapias, Instituto de Biomedicina de Sevilla (IBIS), Avenida Manuel Siurot, s/n, 41013 Seville, Spain. E-mail address: [email protected] (R. Garcia-Carbonero). ☆☆
http://dx.doi.org/10.1016/j.humpath.2014.08.008 0046-8177/© 2014 Elsevier Inc. All rights reserved.
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1. Introduction Colon cancer (CRC) is the third most common cancer worldwide, with 1 361 000 newly diagnosed cases and 694 000 deaths registered in 2012 (http://globocan.iarc.fr). Incidence crude rates are notably higher for men than for women (77.9 versus 58.6 new cases/100 000 habitants/year diagnosed in the European Union in 2012), and a similar sex disbalance is also observed for mortality. Numerous epidemiologic observations and experimental studies suggest a protective effect for estrogens on colon cancer risk. Indeed, controlled clinical trials have shown that hormone replacement therapy reduces the risk of CRC by 30% to 40% in postmenopausal women, and also seem to decrease the incidence and recurrence of adenomatous polyps [1,2]. Moreover, the greater decline observed in mortality rates in aged women compared with men since 1990 has been attributed by some investigators to the increased use of hormone replacement therapy. Based on these observations, one may speculate that endogenous estrogens would also play a protective role and could justify the overall reduced incidence and mortality of CRC observed in women. However, some case-control studies do not support this hypothesis because they have observed a positive association between endogenous estradiol levels and the risk of CRC in postmenopausal women [3,4]. Estrogens regulate growth, differentiation, and function of a variety of tissues, including the gastrointestinal tract [5]. Biological activity of estrogens is mainly exerted through their binding to 2 specific receptors: estrogen receptor α (ERα) and ERβ. Both belong to the nuclear receptor superfamily of transcription factors that are activated upon ligand binding [6]. ER proteins contain 3 major functional domains: a hypervariable N-terminal domain, which contains the ligand-independent transcription activation function 1 (AF-1) region; a highly conserved DNA-binding domain; and a C-terminal domain, which is involved in ligand-binding and ligand-dependent transcription activation functions (AF-2) [7–9]. Upon hormone binding, ERs form homodimers (ERα/ERα, ERβ/ERβ) or heterodimers (ERα/ERβ) [10], which act as ligand-activated transcriptional factors. Estrogens play an important role in the normal development and function of reproductive and nonreproductive tissues. ER subtypes are, however, unevenly distributed among different tissues: ERα is mainly expressed in breast, bone, urogenital tract, cardiovascular tissue, and central nervous system, whereas ERβ is the prevalent form in the gastrointestinal tract [11]. These 2 receptors seem to have opposite roles in the regulation of proliferation and differentiation of target tissues. Preclinical models show that ERβ is able to modulate ERα activity, inhibiting estrogen-dependent proliferation and promoting apoptosis. Consistent with these observations, many lines of evidence suggest a relationship between the perturbation of estrogen signaling and cancer initiation, progression, and response to treatment. More specifically, ERα has been involved in the
I. López-Calderero et al. development and progression of breast cancer and is a useful biomarker to predict response to hormonal therapy. On the contrary, ERβ is the prevalent ER in normal colonic mucosa, whereas its expression is significantly reduced in CRC. Indeed, an increased ERα/β ratio has been documented in colon carcinomas, and this has been associated with increased proliferation and decreased apoptosis [12–18]. Estrogens also modulate gene expression and cell function through a number of alternative mechanisms, including protein phosphorylation and other nongenomic pathways. ERs can be sometimes located at the inner face of the cell membrane, where they may associate with other growth factor receptors such as IGF-1R, EGFR, or HER2. The estrogen-bound membrane ER can induce more rapid effects through the activation of cell signal transduction pathways, such as the mitogen-activated protein kinase and PI3K/AKT pathways. [19,20]. In addition to hormone-dependent activation, ERα function may be modulated by posttranslational modifications such as direct phosphorylation of ERα or coactivators in a ligand-independent manner. The phosphorylated protein forms a more stable transcription complex with the coactivators and up-regulates the transcription activity of the ER, even in the absence of hormone [21]. Ser167, located within the AF1 region of ERα, is a major site of ERα phosphorylation in response to estradiol binding [22,23]. Both AKT and the kinase downstream of mitogen-activated protein kinase, p90 ribosomal S6 kinase (pp90Rsk), can also phosphorylate ERα at Ser167 [14]. Moreover, PI3K may be activated through ERα ligand-dependent binding. As a consequence of PI3K-AKT activation, thus, phosphorylated ERα induces, under estrogen environment, a positive feedback loop that amplifies the signal [23]. Interestingly, retrospective series and randomized clinical trials conducted in patients with breast cancer strongly suggest that pSer118- and/or pSer167-ERα is associated with improved response to hormonal therapy. However, the functional consequences of these complex estrogen signaling pathway regulations need to be further elucidated. Based on these data, the aims of our study were to evaluate pSer167-ERα expression in a cohort of patients with surgically resected stage II-III colon cancer and to assess its potential association with relevant clinicopathological features and its influence on survival.
2. Materials and methods 2.1. Study population Two hundred eighteen consecutive patients with stage IIIII colon cancer attended at our institution from 2000 through 2005 were retrospectively identified to be eligible for this study. Patients had to meet the following inclusion criteria: (1) confirmed histologic diagnosis of primary colon cancer, (2) surgical resection with curative intent, (3) TNM stage II or III, (4) adequate clinical data recorded in medical charts, and
Estrogen receptors and colon cancer prognosis (5) tissue specimen available for additional immunohistochemical assays. Medical records of selected patients were retrospectively reviewed according to a previously designed protocol, which included the following clinical data: sex, age, weight, height, body mass index (BMI), performance status, baseline carcinoembryonic antigen (CEA) levels, baseline plasma glucose levels, history of diabetes, treatment with oral antidiabetic drugs (OADs) and/or insulin, date of diagnosis, date and type of surgery, emergency presentation (bowel obstruction or perforation), tumor location, World Health Organization histologic subtype, tumor differentiation, TNM stage, presence of lymphatic, vascular or perineural invasion, indication of adjuvant chemotherapy (CT), type of CT received, date of relapse, and date of last visit or death. Staging was assessed according to the American Joint Committee on Cancer TNM classification (fifth edition) [24]. Performance status of patients was evaluated following the Eastern Cooperative Oncology Group scale [25]. All procedures were performed in compliance with relevant laws and institutional guidelines, and the corresponding institutional review board approved the study protocol.
2439 and recorded. Tumor samples were also classified according to staining intensity into 3 categories: no staining (0), weak (1+), and moderate (2+) intensity staining (Fig. 1). No tumor sample was found to have strong intensity staining (3+). A phosphoERαSer167 (pERαSer167) score was then calculated with the following formula: higher intensity × % of stained cells. Patients were classified according to this score in 2 groups: low pERαSer167 expression (pERαSer167 score b median) versus high pERαSer167 expression (pERαSer167 score ≥ median; Table 1).
2.3. Statistical analysis All analyses were performed using Statistical Package for the Social Sciences v.16 (SPSS Inc, Chicago, IL). Descriptive statistics were used to characterize the most relevant clinical parameters. The association of categorical clinical and pathophysiologic features and ER expression was assessed by v2 test or Fisher exact test, when
2.2. Immunohistochemistry A total of 218 formalin-fixed, paraffin-embedded tumor samples were provided by the Department of Pathology of our institution. Blocks were assessed by a pathologist for suitability to generate tissue microarray (TMA). Eleven TMAs were constructed with 2 tissue samples of each patient. One section of each TMA was stained with hematoxylin and eosin for histology verification and quality assessment. Each TMA had 3 duplicate control cores of normal colon tissue. Histologic sections (5 μm) of each TMA were stained with pSer167 ERα antibody at 1:25 dilution (#2541; Cell Signaling Technology, Inc, MA) by a pathologist at LVTECH XXI Biotechnology SL (La Rioja, Spain). Briefly, sections were dewaxed in 2 xylene baths (5 minutes each), taken through serial passes of ethanol solutions (100%, 96%, and 70%), rehydrated in distilled water, and then submitted to heat-induced antigen retrieval in the presence of citrate solution (pH 6.0) and phosphatebuffered saline buffer. Blocking of the peroxidase solution was applied for 5 minutes, and then TMA slides were incubated for 30 minutes with protein blocking solution. Finally, primary pSer167 ERα monoclonal antibody was added and a biotinylated antibody was used as a secondary antibody (K0679 15 mL, K0690LSAB+ System-HRP; Dako, Carpinteria, CA). Two independent pathologists blinded to clinical outcome performed the immunohistochemical analyses. TMA sections were scanned at low magnification, and the percentage of positively stained tumor cells was estimated from 0 to 100% of the total of tumor cells. Cellular sublocalization (membrane, cytoplasm, nucleus) of pSer167 ERα expression was assessed
Fig. 1 Immunohistochemistry for pERαSer167 expression in colon cancer. A, Negative expression. B, Weak expression (1+). C, Moderate expression (2+).
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I. López-Calderero et al.
Table 1 Phospho-ERαSer167 expression by immunohistochemical staining Intensity (I) 0 1+ 2+ Cellular location of pERαSer167 staining No staining Nuclear only Nuclear and cytoplasm Cytoplasm only Membranous Percentage of stained cells (%) 0-5 6-10 11-60 ≥60 Phospho-ER αSer167 score (I × %) Median (range) 0-5 5-1-12.5 12.6-60 N60
39 (17.9) 168 (77.1) 11 (5.0) 39 (17.9) 7 (3.1) 13 (6.0) 158 (72.5) 1 (0.5) 25% 25% 25% 25% 12.5 (0-200) 25% 25% 25% 25%
Values are presented as n (%), unless otherwise indicated. Abbreviation: NE, not evaluable.
appropriate. Disease-free interval (DFI) was calculated from the histologic diagnosis of colon cancer to the date of relapse or last visit in the absence of relapse. Overall survival (OS) was calculated from histologic diagnosis to the date of death from any cause or last visit in alive patients. DFI and OS were estimated according to the Kaplan-Meier product-limit method [26], and differences observed among patient subgroups were assessed by the log-rank test [27]. Cox proportional hazards regression model was used to evaluate the prognostic influence of pERαSer167 expression on DFI and OS, while controlling for other potentially confounding factors such as age, sex, glucose levels, history of diabetes, OAD, tumor location, bowel obstruction, baseline CEA levels, T and N stages, and adjuvant CT.
3. Results 3.1. Study population Two hundred eighteen patients were included in the study, 214 of which were evaluable for immunohistochemical assessments (Table 2). Clinicopathological characteristics of the study cohort are detailed in Supplementary Table S1. The median age was 69 years (range, 28-92 years), 57% were male, 66% had stage II tumors, and 43% received adjuvant fluoropyrimidine CT. With a median follow-up of 72 months (range, 0.43-153 months), 51 patients (25%) had relapsed and 67 (31%) had died, 19 of whom (28% of deaths) due to perioperative complications. Overall, DFI and OS at
5 years for the whole series were 71% and 67%, respectively. As expected, DFI was significantly greater for stage II patients than for stage III (78 % versus 58 %, P = .01), and a similar trend was observed for OS (Table 3).
3.2. Correlation of pERα Ser167 expression and clinicopathological features Phospho-ERαSer167 expression was observed in 179 tumor samples (82%). Staining intensity was weak (1+) in 168 (77%) cases and moderate (2+) in 11 (5%). Staining was nuclear in 9% of tumor samples, cytoplasmic in 73%, and membranous in only 1 case. Half of the samples had positive staining in more than 10% of tumor cells. Median pERαSer167 score was 12.5 (range, 0-200; Table 1). The associations observed between pERαSer167 expression and different clinicopathological features are summarized in Table 3. High pERαSer167 score was more commonly observed in women versus men (56% versus 45%; P = .066), older (N65 years) versus younger patients (40% versus 56%; P = .018), patients with baseline glucose levels higher than 120 mg/dL versus 120 mg/dL or lower (45% versus 66%; P = .013), patients treated with OADs versus nontreated (68% versus 45%; P = .020), and in those who had received adjuvant CT versus those who had not (59% versus 49%; P = .019).
3.3. Impact of pERα Ser167 expression on survival The potential impact of different clinicopathological features on DFI and OS was first assessed by univariate analysis. As shown in Table 3, right colon tumor location (P = .002), T4 (P = .045), metastatic lymph nodes (P = .010), poor differentiation (P = .019), and stage III disease (P = .010) were features significantly associated with a shorter DFI. Moreover, age over 65 years (P = .004), high baseline CEA levels (P = .019), T4 (P = .064), and no adjuvant CT (P = .001) were all associated with a significantly worse OS. Regarding pERαSer167 expression, 5-year DFI was lower for patients with high versus low pERαSer167 score (≥median versus bmedian), and the observed difference was of borderline significance (66% versus 78%%; P = .066). Of note, a trend toward worse prognostic implications for nuclear staining versus cytoplasmatic staining was documented, although the observed differences were not statistically significant (DFI at 5 years: 81% for negative staining, 71% for cytoplasmic staining, and 65% for nuclear staining; P = .483). Similarly, patients with a high pERαSer167 score were found to have a shorter OS than those with a low score (65% versus 73% alive at 5 years; P = .462; Table 4). Tumors with nuclear pERαSer167 expression tended to have lower 5-year DFI (65% versus 71%, P = .722) and OS rates (51% versus 68%, P = .871) than did those with cytoplasmic staining. In exploratory analysis, a high pERαSer167 score was significantly associated with a worse 5-year DFI in the following subgroups: women (85% versus
Estrogen receptors and colon cancer prognosis Table 2
2441
Study population characteristics and association with pERαSer167 expression n (%)
Low Sex Male Female Age (y) ≤65 N65 ECOG PS a 0 1 2 BMI a Nonobese (b30 kg/m2) Obese (≥30 kg/m2) Type 2 diabetes a Yes No Glucose levels a ≤120 mg/dL N120 mg/dL Oral antidiabetic agents a Yes No CEA a Normal High Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T T3 T4 N N0 N1 N2 Tumor grade a G1 G2 G3 TNM stage II III Adjuvant CT a Yes No
v2, P
pERαser167score, n (%) High
.066 124 (57) 94 (43)
68 (55) 41 (44)
56 (45) 53 (56)
82 (38) 136 (62)
49 (60) 60 (44)
33 (40) 79 (56)
157 (72) 18 (8) 6 (3)
77 (49) 5 (27) 4 (67)
80 (51) 13 (73) 2 (33)
109 (50) 50 (23)
57 (52) 23 (46)
52 (47) 27 (54)
40 (18) 141 (65)
17 (43) 74 (52)
23 (47) 67 (48)
137 (63) 41 (19)
76 (55) 14 (34)
61 (45) 27 (66)
31 (14) 143 (66)
10 (32) 78 (55)
21 (68) 65 (45)
52 (24) 60 (27)
29 (56) 32 (53)
23 (44) 28 (47)
56 (26) 162 (74)
30 (53) 79 (49)
27 (47) 82 (51)
45 (21) 173 (79)
26 (58) 83 (48)
19 (42) 90 (52)
16 (7) 202 (93)
9 (56) 100 (50)
7 (44) 102 (50)
189 (87) 25 (11)
92 (49) 14 (56)
97 (51) 11 (44)
145 (66) 56 (26) 17 (8)
71 (49) 30 (54) 9 (56)
74 (51) 26 (46) 8 (47)
98 (45) 86 (40) 19 (9)
50 (51) 38 (44) 13 (68)
48 (49) 48 (56) 6 (32)
145 (67) 73 (33)
70 (49) 39 (53)
75 (51) 34 (47)
88 (40) 128 (59)
36 (41) 72 (56)
52 (59) 56 (44)
.018 ⁎
.222
.286
.175 .013 ⁎ .020 ⁎
.386
.379
.158
.398
.317
.798
.225
.299 .019 ⁎
Abbreviations: BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; ECOG PS, Eastern Cooperative Oncology Group performance status; T, tumor size, N, lymph node metastasis; M, distant metastasis. a Parameters that include fewer total cases due to missing values (see Supplementary Table S1 for detailed information). ⁎ Statistically significant.
2442 Table 3
I. López-Calderero et al. DFI and OS according to clinicopathological features
Sex Male Female Age (y) ≤65 N65 ECOG PS a 0 1 ≥2 BMI a Nonobese Obese (≥30 kg/m2) Type 2 diabetes a No Yes Glucose levels a ≤120 mg/dL N120 mg/dL Oral antidiabetics a Yes No Baseline CEA a Normal High-UK Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T pT1-T3 pT4 N pN0 pN1-2 Tumor grade a G1 Other TNM stage II III Adjuvant CT a Yes No
n (%)
DFI (% at 5 y)
124 (57%) 94 (43%)
73% 71%
82 (38%) 136 (62%)
73% 71%
157 (72%) 18 (8%) 9 (4%)
74% 67% 60%
109 (50%) 50 (23%)
78% 72%
141 (65%) 40 (18%)
76% 75%
137 (63%) 41 (19%)
77% 75%
31 (14%) 143 (66%)
75% 82%
P
OS (% at 5 y)
.754
.840 71% 71%
.981
.638 81% 75% 33%
.458
.678 79% 83%
.946
.643 75% 72%
.819
.873 76% 76%
.321
.823 74% 78%
.147 81% 68%
56 (26%) 162 (74%)
56% 78%
45 (21%) 173 (79%)
60% 74%
16 (7%) 202 (93%)
60% 73%
193 (89%) 25 (11%)
73% 55%
144 (67%) 73 (33%)
78% 58%
98 (45%) 110 (55%)
79% 66%
144 (67%) 73 (33%)
78% 58%
88 (40%) 128 (59%)
66% 77%
.004 ⁎
85% 63% .638
52 (24%) 166 (76%)
P
.002 ⁎
81% 68% 59% 76%
.135
.019 ⁎ .209
.088 ⁎
63% 74% .384 .045 ⁎ .010 ⁎ .019 ⁎ .010 ⁎
.117 55% 73%
.046 ⁎
74% 71% .624 73% 71% .743 74% 71% .624 73% 71%
.076
.001 ⁎
87% 63%
Abbreviations: DFI, disease-free interval; OS, overall survival; ECOG PS, Eastern Cooperative Oncology Group performance status; BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; T, tumor size; N, lymph node metastasis; M, distant metastasis. a Parameters that include fewer total cases due to missing values (see Table S1 for detailed information). ⁎ Statistically significant.
60%; P = .026), young (82% versus 61%; P = .019), obese (89% versus 60%; P = .050), nondiabetic (85% versus 66%; P = .008), low glucose levels (85% versus 66%; P = .015), no
oral antidiabetic treatment (82% versus 69%; P = .039), bowel obstruction (76% versus 31% P = .007), tumor location other than the right colon (87% versus 70%; P = .040), N0 (86%
Estrogen receptors and colon cancer prognosis Table 4
2443
Kaplan-Meier survival analysis according to pERαSer167 expression
pERαSer167 score All patients Sex Male Female Age (y) ≤65 N65 ECOG PS 0 1 2 BMI Nonobese Obese Type 2 diabetes Yes No Glucose levels ≤120 mg/dL N120 mg/dL Oral antidiabetics Yes No CEA Normal High Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T T3 T4 N N0 N1-2 Tumor grade I NI TNM stage II III ECOG PS 0 1 2 Adjuvant CT Yes No
DFI (% at 5 y), ≤medianN
P
OS (% at 5 y), ≤medianN
P
78%-66%
.066 ⁎ .080 ⁎ .723 .026 ⁎ .062 ⁎ .019 ⁎ .660 .115 .299 .087 .886 .038 ⁎ .250 .050 ⁎ .048 ⁎ .372 .008 ⁎ .018 ⁎ .015 ⁎ .670 .021 ⁎ .242 .039 ⁎ .097 ⁎ .401 .142 .059 ⁎ .894 .017 ⁎ .026 ⁎ .007 ⁎ .271 .060 ⁎ .053 ⁎ .012 ⁎ .051 ⁎ .151 .110 .070 ⁎ .040 ⁎ .448 .071 ⁎ .082 ⁎ .316 .058 ⁎ .040 ⁎ .557 .115 .299 .087 .886 .141 .275 .312
73%-65%
.462 .456 .794 .144 .794 .459 .879 .227 .296 .144 .157 .634 .900 .315 .592 .092 .139 .477 .465 .899 .477 .138 .169 .738 .690 .489 .406 .276 .787 .314 .009 ⁎ .758 .406 .405 .246 .451 .255 .488 .524 .343 .811 .533 .324 .954 .524 .343 .811 .227 .296 .144 .157 .208 .138 .575
73%-73% 85%-60% 82%-61% 74%-69% 78%-71% NE-53% 67%-50% 82%-74% 89%-60% 69%-79% 85%-66% 85%-66% 83%-72% NE-76% 82%-69% 84%-78% 83%-63% 57%-54% 87%-70% 76%-31% 79%-71% 33%-NE 81%-85% 78%-69% 71%-33% 86%-72% 62%-53% 87%-72% 74%-61% 86%-72% 62%-53% 78%-71% NE-53% 67%-50% 72%-62% 82%-72%
69%-74% 80%-66% 85%-81% 62%-66% 82%-80% NE-64% 0%-50% 80-76% 83-80% 55%-84% 81%-69% 78%-74% 79%-75% 56%-82% 81%-75% 82%-90% 71%-66% 66%-51% 76%-77% 80%-34% 71%-78% 44%-69% 76%-71% 77%-72% 47%-66% 74%-72% 71%-70% 79%-69% 68%-71% 74%-72% 71%-70% 82%-80% NE-64% 0%-50% 89%-84% 65%-59%
Abbreviations: DFI, disease-free interval; OS, overall survival; ECOG PS, Eastern Cooperative Oncology Group performance status; BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; T, tumor size; N, lymph node metastasis; M, distant metastasis. ⁎ Statistically significant.
Time (months)
P = ,019
Fig. 2 DFI by pERα expression (low expression: pERα score b median; high expression: pERα B, Female. C, Nondiabetic patients. D, Younger patients (≤65 years old). E, Obese patients (≥30 kg/m2). Ser167
Time (months) Time (months)
Ser167
Time (months)
Ser167
D P = ,008
C A
P = ,066
B
P = ,026
YOUNGER PATIENTS (
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Original contribution
Prognostic relevance of estrogen receptor-α Ser167 phosphorylation in stage II-III colon cancer patients☆,☆☆ Iker López-Calderero MD a,b , Amancio Carnero PhD a , Aurora Astudillo MD, PhD c , José Palacios MD, PhD d , Manuel Chaves MD b , Marta Benavent MD a,b , María L. Limón MD b , Rocio Garcia-Carbonero MD, PhD a,b,⁎ a
Instituto de Biomedicina de Sevilla (IBIS), HUVR/CSIC/Universidad de Sevilla, Center affiliated to the Red Temática de Investigación Cooperativa en Cancer (RTICC), Instituto Carlos III, Spanish Ministry of Science and Innovation, Seville, 41013, Spain b Department of Medical Oncology, Hospital Universitario Virgen del Rocío, Center affiliated to the Red Temática de Investigación Cooperativa en Cancer (RTICC), Instituto Carlos III, Spanish Ministry of Science and Innovation, Seville, 41013, Spain c Department of Pathology, Hospital Central de Asturias, Asturias, 33006, Spain d Department of Pathology, Hospital Universitario Virgen del Rocío, Seville, 41013, Spain Received 2 June 2014; revised 22 July 2014; accepted 5 August 2014
Keywords: Colon cancer; Tissue microarray; Estrogen receptor; Immunohistochemistry; Prognosis; Survival; Biomarkers
Summary Preclinical and clinical data suggest a protective role for estrogens on colon cancer (CRC) risk. estrogen receptor (ER) β is the prevalent ER in normal colonic mucosa, whereas its expression is significantly reduced in CRC. An increased ERα/β ratio has been documented in colon carcinomas and is associated with increased proliferation and decreased apoptosis. The aim of our study was to evaluate the expression of activated ERα and its prognostic implications in patients with stage II-III CRC. Phospho-ERαSer167 (pERαSer167) expression was assessed by immunohistochemistry in 218 CRC paraffin-embedded tumor samples. A high pERαSer167 expression was more commonly observed in women, older patients, and patients with high baseline glucose levels. This higher pERαSer167 expression was associated with decreased 5-year disease-free interval (DFI; 66% versus 78%, P = .07) and overall survival (65% versus 73%, P = .46). The negative impact of high pERαSer167 expression on DFI was particularly significant (P b .05) in women (85% versus 60%), young (82% versus 61%), nondiabetic (85% versus 66%), and stage II patients (86% versus 72% and low versus high pERαSer167, respectively). Multivariate analysis confirmed that pERαSer167 score was a significant prognostic factor for both DFI and overall survival, independent of sex, age, glucose levels, tumor stage, bowel obstruction/ perforation, or adjuvant chemotherapy. These findings illustrate the relevance of estrogen pathways in colon cancer biology and may provide novel therapeutic avenues to be explored in this context. © 2014 Elsevier Inc. All rights reserved.
☆
Competing interests: The authors declare no conflicts of interest. Funding/Support: This work was partially supported by a grant from the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III Spanish Cancer Networks RTICC (R12/0036/0008 and R12/0036/0028). R.G.C. is funded by the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (PI10/02164, PI13/02295), Servicio Andaluz de Salud (PI-0259/2007), and Fundacion Mutua Madrileña (P0497/2006). A.C. is funded by the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (FIS PI12/00137), Consejeria de Ciencia e Innovacion (CTS-6844, CM09/00207), and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). ⁎ Corresponding author at: Medical Oncology Department, Hospital Universitario Virgen del Rocio, Laboratorio de Oncologia Traslacional y Nuevas Terapias, Instituto de Biomedicina de Sevilla (IBIS), Avenida Manuel Siurot, s/n, 41013 Seville, Spain. E-mail address: [email protected] (R. Garcia-Carbonero). ☆☆
http://dx.doi.org/10.1016/j.humpath.2014.08.008 0046-8177/© 2014 Elsevier Inc. All rights reserved.
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1. Introduction Colon cancer (CRC) is the third most common cancer worldwide, with 1 361 000 newly diagnosed cases and 694 000 deaths registered in 2012 (http://globocan.iarc.fr). Incidence crude rates are notably higher for men than for women (77.9 versus 58.6 new cases/100 000 habitants/year diagnosed in the European Union in 2012), and a similar sex disbalance is also observed for mortality. Numerous epidemiologic observations and experimental studies suggest a protective effect for estrogens on colon cancer risk. Indeed, controlled clinical trials have shown that hormone replacement therapy reduces the risk of CRC by 30% to 40% in postmenopausal women, and also seem to decrease the incidence and recurrence of adenomatous polyps [1,2]. Moreover, the greater decline observed in mortality rates in aged women compared with men since 1990 has been attributed by some investigators to the increased use of hormone replacement therapy. Based on these observations, one may speculate that endogenous estrogens would also play a protective role and could justify the overall reduced incidence and mortality of CRC observed in women. However, some case-control studies do not support this hypothesis because they have observed a positive association between endogenous estradiol levels and the risk of CRC in postmenopausal women [3,4]. Estrogens regulate growth, differentiation, and function of a variety of tissues, including the gastrointestinal tract [5]. Biological activity of estrogens is mainly exerted through their binding to 2 specific receptors: estrogen receptor α (ERα) and ERβ. Both belong to the nuclear receptor superfamily of transcription factors that are activated upon ligand binding [6]. ER proteins contain 3 major functional domains: a hypervariable N-terminal domain, which contains the ligand-independent transcription activation function 1 (AF-1) region; a highly conserved DNA-binding domain; and a C-terminal domain, which is involved in ligand-binding and ligand-dependent transcription activation functions (AF-2) [7–9]. Upon hormone binding, ERs form homodimers (ERα/ERα, ERβ/ERβ) or heterodimers (ERα/ERβ) [10], which act as ligand-activated transcriptional factors. Estrogens play an important role in the normal development and function of reproductive and nonreproductive tissues. ER subtypes are, however, unevenly distributed among different tissues: ERα is mainly expressed in breast, bone, urogenital tract, cardiovascular tissue, and central nervous system, whereas ERβ is the prevalent form in the gastrointestinal tract [11]. These 2 receptors seem to have opposite roles in the regulation of proliferation and differentiation of target tissues. Preclinical models show that ERβ is able to modulate ERα activity, inhibiting estrogen-dependent proliferation and promoting apoptosis. Consistent with these observations, many lines of evidence suggest a relationship between the perturbation of estrogen signaling and cancer initiation, progression, and response to treatment. More specifically, ERα has been involved in the
I. López-Calderero et al. development and progression of breast cancer and is a useful biomarker to predict response to hormonal therapy. On the contrary, ERβ is the prevalent ER in normal colonic mucosa, whereas its expression is significantly reduced in CRC. Indeed, an increased ERα/β ratio has been documented in colon carcinomas, and this has been associated with increased proliferation and decreased apoptosis [12–18]. Estrogens also modulate gene expression and cell function through a number of alternative mechanisms, including protein phosphorylation and other nongenomic pathways. ERs can be sometimes located at the inner face of the cell membrane, where they may associate with other growth factor receptors such as IGF-1R, EGFR, or HER2. The estrogen-bound membrane ER can induce more rapid effects through the activation of cell signal transduction pathways, such as the mitogen-activated protein kinase and PI3K/AKT pathways. [19,20]. In addition to hormone-dependent activation, ERα function may be modulated by posttranslational modifications such as direct phosphorylation of ERα or coactivators in a ligand-independent manner. The phosphorylated protein forms a more stable transcription complex with the coactivators and up-regulates the transcription activity of the ER, even in the absence of hormone [21]. Ser167, located within the AF1 region of ERα, is a major site of ERα phosphorylation in response to estradiol binding [22,23]. Both AKT and the kinase downstream of mitogen-activated protein kinase, p90 ribosomal S6 kinase (pp90Rsk), can also phosphorylate ERα at Ser167 [14]. Moreover, PI3K may be activated through ERα ligand-dependent binding. As a consequence of PI3K-AKT activation, thus, phosphorylated ERα induces, under estrogen environment, a positive feedback loop that amplifies the signal [23]. Interestingly, retrospective series and randomized clinical trials conducted in patients with breast cancer strongly suggest that pSer118- and/or pSer167-ERα is associated with improved response to hormonal therapy. However, the functional consequences of these complex estrogen signaling pathway regulations need to be further elucidated. Based on these data, the aims of our study were to evaluate pSer167-ERα expression in a cohort of patients with surgically resected stage II-III colon cancer and to assess its potential association with relevant clinicopathological features and its influence on survival.
2. Materials and methods 2.1. Study population Two hundred eighteen consecutive patients with stage IIIII colon cancer attended at our institution from 2000 through 2005 were retrospectively identified to be eligible for this study. Patients had to meet the following inclusion criteria: (1) confirmed histologic diagnosis of primary colon cancer, (2) surgical resection with curative intent, (3) TNM stage II or III, (4) adequate clinical data recorded in medical charts, and
Estrogen receptors and colon cancer prognosis (5) tissue specimen available for additional immunohistochemical assays. Medical records of selected patients were retrospectively reviewed according to a previously designed protocol, which included the following clinical data: sex, age, weight, height, body mass index (BMI), performance status, baseline carcinoembryonic antigen (CEA) levels, baseline plasma glucose levels, history of diabetes, treatment with oral antidiabetic drugs (OADs) and/or insulin, date of diagnosis, date and type of surgery, emergency presentation (bowel obstruction or perforation), tumor location, World Health Organization histologic subtype, tumor differentiation, TNM stage, presence of lymphatic, vascular or perineural invasion, indication of adjuvant chemotherapy (CT), type of CT received, date of relapse, and date of last visit or death. Staging was assessed according to the American Joint Committee on Cancer TNM classification (fifth edition) [24]. Performance status of patients was evaluated following the Eastern Cooperative Oncology Group scale [25]. All procedures were performed in compliance with relevant laws and institutional guidelines, and the corresponding institutional review board approved the study protocol.
2439 and recorded. Tumor samples were also classified according to staining intensity into 3 categories: no staining (0), weak (1+), and moderate (2+) intensity staining (Fig. 1). No tumor sample was found to have strong intensity staining (3+). A phosphoERαSer167 (pERαSer167) score was then calculated with the following formula: higher intensity × % of stained cells. Patients were classified according to this score in 2 groups: low pERαSer167 expression (pERαSer167 score b median) versus high pERαSer167 expression (pERαSer167 score ≥ median; Table 1).
2.3. Statistical analysis All analyses were performed using Statistical Package for the Social Sciences v.16 (SPSS Inc, Chicago, IL). Descriptive statistics were used to characterize the most relevant clinical parameters. The association of categorical clinical and pathophysiologic features and ER expression was assessed by v2 test or Fisher exact test, when
2.2. Immunohistochemistry A total of 218 formalin-fixed, paraffin-embedded tumor samples were provided by the Department of Pathology of our institution. Blocks were assessed by a pathologist for suitability to generate tissue microarray (TMA). Eleven TMAs were constructed with 2 tissue samples of each patient. One section of each TMA was stained with hematoxylin and eosin for histology verification and quality assessment. Each TMA had 3 duplicate control cores of normal colon tissue. Histologic sections (5 μm) of each TMA were stained with pSer167 ERα antibody at 1:25 dilution (#2541; Cell Signaling Technology, Inc, MA) by a pathologist at LVTECH XXI Biotechnology SL (La Rioja, Spain). Briefly, sections were dewaxed in 2 xylene baths (5 minutes each), taken through serial passes of ethanol solutions (100%, 96%, and 70%), rehydrated in distilled water, and then submitted to heat-induced antigen retrieval in the presence of citrate solution (pH 6.0) and phosphatebuffered saline buffer. Blocking of the peroxidase solution was applied for 5 minutes, and then TMA slides were incubated for 30 minutes with protein blocking solution. Finally, primary pSer167 ERα monoclonal antibody was added and a biotinylated antibody was used as a secondary antibody (K0679 15 mL, K0690LSAB+ System-HRP; Dako, Carpinteria, CA). Two independent pathologists blinded to clinical outcome performed the immunohistochemical analyses. TMA sections were scanned at low magnification, and the percentage of positively stained tumor cells was estimated from 0 to 100% of the total of tumor cells. Cellular sublocalization (membrane, cytoplasm, nucleus) of pSer167 ERα expression was assessed
Fig. 1 Immunohistochemistry for pERαSer167 expression in colon cancer. A, Negative expression. B, Weak expression (1+). C, Moderate expression (2+).
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I. López-Calderero et al.
Table 1 Phospho-ERαSer167 expression by immunohistochemical staining Intensity (I) 0 1+ 2+ Cellular location of pERαSer167 staining No staining Nuclear only Nuclear and cytoplasm Cytoplasm only Membranous Percentage of stained cells (%) 0-5 6-10 11-60 ≥60 Phospho-ER αSer167 score (I × %) Median (range) 0-5 5-1-12.5 12.6-60 N60
39 (17.9) 168 (77.1) 11 (5.0) 39 (17.9) 7 (3.1) 13 (6.0) 158 (72.5) 1 (0.5) 25% 25% 25% 25% 12.5 (0-200) 25% 25% 25% 25%
Values are presented as n (%), unless otherwise indicated. Abbreviation: NE, not evaluable.
appropriate. Disease-free interval (DFI) was calculated from the histologic diagnosis of colon cancer to the date of relapse or last visit in the absence of relapse. Overall survival (OS) was calculated from histologic diagnosis to the date of death from any cause or last visit in alive patients. DFI and OS were estimated according to the Kaplan-Meier product-limit method [26], and differences observed among patient subgroups were assessed by the log-rank test [27]. Cox proportional hazards regression model was used to evaluate the prognostic influence of pERαSer167 expression on DFI and OS, while controlling for other potentially confounding factors such as age, sex, glucose levels, history of diabetes, OAD, tumor location, bowel obstruction, baseline CEA levels, T and N stages, and adjuvant CT.
3. Results 3.1. Study population Two hundred eighteen patients were included in the study, 214 of which were evaluable for immunohistochemical assessments (Table 2). Clinicopathological characteristics of the study cohort are detailed in Supplementary Table S1. The median age was 69 years (range, 28-92 years), 57% were male, 66% had stage II tumors, and 43% received adjuvant fluoropyrimidine CT. With a median follow-up of 72 months (range, 0.43-153 months), 51 patients (25%) had relapsed and 67 (31%) had died, 19 of whom (28% of deaths) due to perioperative complications. Overall, DFI and OS at
5 years for the whole series were 71% and 67%, respectively. As expected, DFI was significantly greater for stage II patients than for stage III (78 % versus 58 %, P = .01), and a similar trend was observed for OS (Table 3).
3.2. Correlation of pERα Ser167 expression and clinicopathological features Phospho-ERαSer167 expression was observed in 179 tumor samples (82%). Staining intensity was weak (1+) in 168 (77%) cases and moderate (2+) in 11 (5%). Staining was nuclear in 9% of tumor samples, cytoplasmic in 73%, and membranous in only 1 case. Half of the samples had positive staining in more than 10% of tumor cells. Median pERαSer167 score was 12.5 (range, 0-200; Table 1). The associations observed between pERαSer167 expression and different clinicopathological features are summarized in Table 3. High pERαSer167 score was more commonly observed in women versus men (56% versus 45%; P = .066), older (N65 years) versus younger patients (40% versus 56%; P = .018), patients with baseline glucose levels higher than 120 mg/dL versus 120 mg/dL or lower (45% versus 66%; P = .013), patients treated with OADs versus nontreated (68% versus 45%; P = .020), and in those who had received adjuvant CT versus those who had not (59% versus 49%; P = .019).
3.3. Impact of pERα Ser167 expression on survival The potential impact of different clinicopathological features on DFI and OS was first assessed by univariate analysis. As shown in Table 3, right colon tumor location (P = .002), T4 (P = .045), metastatic lymph nodes (P = .010), poor differentiation (P = .019), and stage III disease (P = .010) were features significantly associated with a shorter DFI. Moreover, age over 65 years (P = .004), high baseline CEA levels (P = .019), T4 (P = .064), and no adjuvant CT (P = .001) were all associated with a significantly worse OS. Regarding pERαSer167 expression, 5-year DFI was lower for patients with high versus low pERαSer167 score (≥median versus bmedian), and the observed difference was of borderline significance (66% versus 78%%; P = .066). Of note, a trend toward worse prognostic implications for nuclear staining versus cytoplasmatic staining was documented, although the observed differences were not statistically significant (DFI at 5 years: 81% for negative staining, 71% for cytoplasmic staining, and 65% for nuclear staining; P = .483). Similarly, patients with a high pERαSer167 score were found to have a shorter OS than those with a low score (65% versus 73% alive at 5 years; P = .462; Table 4). Tumors with nuclear pERαSer167 expression tended to have lower 5-year DFI (65% versus 71%, P = .722) and OS rates (51% versus 68%, P = .871) than did those with cytoplasmic staining. In exploratory analysis, a high pERαSer167 score was significantly associated with a worse 5-year DFI in the following subgroups: women (85% versus
Estrogen receptors and colon cancer prognosis Table 2
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Study population characteristics and association with pERαSer167 expression n (%)
Low Sex Male Female Age (y) ≤65 N65 ECOG PS a 0 1 2 BMI a Nonobese (b30 kg/m2) Obese (≥30 kg/m2) Type 2 diabetes a Yes No Glucose levels a ≤120 mg/dL N120 mg/dL Oral antidiabetic agents a Yes No CEA a Normal High Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T T3 T4 N N0 N1 N2 Tumor grade a G1 G2 G3 TNM stage II III Adjuvant CT a Yes No
v2, P
pERαser167score, n (%) High
.066 124 (57) 94 (43)
68 (55) 41 (44)
56 (45) 53 (56)
82 (38) 136 (62)
49 (60) 60 (44)
33 (40) 79 (56)
157 (72) 18 (8) 6 (3)
77 (49) 5 (27) 4 (67)
80 (51) 13 (73) 2 (33)
109 (50) 50 (23)
57 (52) 23 (46)
52 (47) 27 (54)
40 (18) 141 (65)
17 (43) 74 (52)
23 (47) 67 (48)
137 (63) 41 (19)
76 (55) 14 (34)
61 (45) 27 (66)
31 (14) 143 (66)
10 (32) 78 (55)
21 (68) 65 (45)
52 (24) 60 (27)
29 (56) 32 (53)
23 (44) 28 (47)
56 (26) 162 (74)
30 (53) 79 (49)
27 (47) 82 (51)
45 (21) 173 (79)
26 (58) 83 (48)
19 (42) 90 (52)
16 (7) 202 (93)
9 (56) 100 (50)
7 (44) 102 (50)
189 (87) 25 (11)
92 (49) 14 (56)
97 (51) 11 (44)
145 (66) 56 (26) 17 (8)
71 (49) 30 (54) 9 (56)
74 (51) 26 (46) 8 (47)
98 (45) 86 (40) 19 (9)
50 (51) 38 (44) 13 (68)
48 (49) 48 (56) 6 (32)
145 (67) 73 (33)
70 (49) 39 (53)
75 (51) 34 (47)
88 (40) 128 (59)
36 (41) 72 (56)
52 (59) 56 (44)
.018 ⁎
.222
.286
.175 .013 ⁎ .020 ⁎
.386
.379
.158
.398
.317
.798
.225
.299 .019 ⁎
Abbreviations: BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; ECOG PS, Eastern Cooperative Oncology Group performance status; T, tumor size, N, lymph node metastasis; M, distant metastasis. a Parameters that include fewer total cases due to missing values (see Supplementary Table S1 for detailed information). ⁎ Statistically significant.
2442 Table 3
I. López-Calderero et al. DFI and OS according to clinicopathological features
Sex Male Female Age (y) ≤65 N65 ECOG PS a 0 1 ≥2 BMI a Nonobese Obese (≥30 kg/m2) Type 2 diabetes a No Yes Glucose levels a ≤120 mg/dL N120 mg/dL Oral antidiabetics a Yes No Baseline CEA a Normal High-UK Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T pT1-T3 pT4 N pN0 pN1-2 Tumor grade a G1 Other TNM stage II III Adjuvant CT a Yes No
n (%)
DFI (% at 5 y)
124 (57%) 94 (43%)
73% 71%
82 (38%) 136 (62%)
73% 71%
157 (72%) 18 (8%) 9 (4%)
74% 67% 60%
109 (50%) 50 (23%)
78% 72%
141 (65%) 40 (18%)
76% 75%
137 (63%) 41 (19%)
77% 75%
31 (14%) 143 (66%)
75% 82%
P
OS (% at 5 y)
.754
.840 71% 71%
.981
.638 81% 75% 33%
.458
.678 79% 83%
.946
.643 75% 72%
.819
.873 76% 76%
.321
.823 74% 78%
.147 81% 68%
56 (26%) 162 (74%)
56% 78%
45 (21%) 173 (79%)
60% 74%
16 (7%) 202 (93%)
60% 73%
193 (89%) 25 (11%)
73% 55%
144 (67%) 73 (33%)
78% 58%
98 (45%) 110 (55%)
79% 66%
144 (67%) 73 (33%)
78% 58%
88 (40%) 128 (59%)
66% 77%
.004 ⁎
85% 63% .638
52 (24%) 166 (76%)
P
.002 ⁎
81% 68% 59% 76%
.135
.019 ⁎ .209
.088 ⁎
63% 74% .384 .045 ⁎ .010 ⁎ .019 ⁎ .010 ⁎
.117 55% 73%
.046 ⁎
74% 71% .624 73% 71% .743 74% 71% .624 73% 71%
.076
.001 ⁎
87% 63%
Abbreviations: DFI, disease-free interval; OS, overall survival; ECOG PS, Eastern Cooperative Oncology Group performance status; BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; T, tumor size; N, lymph node metastasis; M, distant metastasis. a Parameters that include fewer total cases due to missing values (see Table S1 for detailed information). ⁎ Statistically significant.
60%; P = .026), young (82% versus 61%; P = .019), obese (89% versus 60%; P = .050), nondiabetic (85% versus 66%; P = .008), low glucose levels (85% versus 66%; P = .015), no
oral antidiabetic treatment (82% versus 69%; P = .039), bowel obstruction (76% versus 31% P = .007), tumor location other than the right colon (87% versus 70%; P = .040), N0 (86%
Estrogen receptors and colon cancer prognosis Table 4
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Kaplan-Meier survival analysis according to pERαSer167 expression
pERαSer167 score All patients Sex Male Female Age (y) ≤65 N65 ECOG PS 0 1 2 BMI Nonobese Obese Type 2 diabetes Yes No Glucose levels ≤120 mg/dL N120 mg/dL Oral antidiabetics Yes No CEA Normal High Tumor location Right colon Other Bowel obstruction Yes No Bowel perforation Yes No T T3 T4 N N0 N1-2 Tumor grade I NI TNM stage II III ECOG PS 0 1 2 Adjuvant CT Yes No
DFI (% at 5 y), ≤medianN
P
OS (% at 5 y), ≤medianN
P
78%-66%
.066 ⁎ .080 ⁎ .723 .026 ⁎ .062 ⁎ .019 ⁎ .660 .115 .299 .087 .886 .038 ⁎ .250 .050 ⁎ .048 ⁎ .372 .008 ⁎ .018 ⁎ .015 ⁎ .670 .021 ⁎ .242 .039 ⁎ .097 ⁎ .401 .142 .059 ⁎ .894 .017 ⁎ .026 ⁎ .007 ⁎ .271 .060 ⁎ .053 ⁎ .012 ⁎ .051 ⁎ .151 .110 .070 ⁎ .040 ⁎ .448 .071 ⁎ .082 ⁎ .316 .058 ⁎ .040 ⁎ .557 .115 .299 .087 .886 .141 .275 .312
73%-65%
.462 .456 .794 .144 .794 .459 .879 .227 .296 .144 .157 .634 .900 .315 .592 .092 .139 .477 .465 .899 .477 .138 .169 .738 .690 .489 .406 .276 .787 .314 .009 ⁎ .758 .406 .405 .246 .451 .255 .488 .524 .343 .811 .533 .324 .954 .524 .343 .811 .227 .296 .144 .157 .208 .138 .575
73%-73% 85%-60% 82%-61% 74%-69% 78%-71% NE-53% 67%-50% 82%-74% 89%-60% 69%-79% 85%-66% 85%-66% 83%-72% NE-76% 82%-69% 84%-78% 83%-63% 57%-54% 87%-70% 76%-31% 79%-71% 33%-NE 81%-85% 78%-69% 71%-33% 86%-72% 62%-53% 87%-72% 74%-61% 86%-72% 62%-53% 78%-71% NE-53% 67%-50% 72%-62% 82%-72%
69%-74% 80%-66% 85%-81% 62%-66% 82%-80% NE-64% 0%-50% 80-76% 83-80% 55%-84% 81%-69% 78%-74% 79%-75% 56%-82% 81%-75% 82%-90% 71%-66% 66%-51% 76%-77% 80%-34% 71%-78% 44%-69% 76%-71% 77%-72% 47%-66% 74%-72% 71%-70% 79%-69% 68%-71% 74%-72% 71%-70% 82%-80% NE-64% 0%-50% 89%-84% 65%-59%
Abbreviations: DFI, disease-free interval; OS, overall survival; ECOG PS, Eastern Cooperative Oncology Group performance status; BMI, body mass index; CEA, carcinoembryonic antigen; CT, chemotherapy; T, tumor size; N, lymph node metastasis; M, distant metastasis. ⁎ Statistically significant.
Time (months)
P = ,019
Fig. 2 DFI by pERα expression (low expression: pERα score b median; high expression: pERα B, Female. C, Nondiabetic patients. D, Younger patients (≤65 years old). E, Obese patients (≥30 kg/m2). Ser167
Time (months) Time (months)
Ser167
Time (months)
Ser167
D P = ,008
C A
P = ,066
B
P = ,026
YOUNGER PATIENTS (
