Glut-1 Expression Correlates with Basal-like Breast Cancer Academic research paper on "Clinical medicine"

Abstract

INTRODUCTION: Glucose transporter 1 (Glut-1) is a facilitative glucose transporter expressed in many cancers including breast cancer. Basal-like breast cancer (BLBC) is a high-risk disease associated with poor prognosis and lacks the benefit of targeted therapy. The aim of this study was to characterize the immunohistochemical (IHC) expression of Glut-1 in patients with BLBC compared with non-BLBC. MATERIALS AND METHODS: We identified 523 cases of invasive breast carcinoma from our database. The clinicopathologic findings and the biologic markers including estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2) status were reviewed. IHC stains for cytokeratin 5/6 (CK5/6), epidermal growth factor receptor (EGFR), p53, and Glut-1 were performed on tissue microarray using standard procedures. BLBC was defined as ER-, PR-, Her2-, and CK5/6+ and/or EGFR+. RESULTS: Of informative cases, 14.7% were categorized as BLBC versus 85.3% as non-BLBC. Glut-1 was expressed in 42 (76.4%) of 55 BLBCs, whereas only 55 (23.8%) of 231 non-BLBCs showed immunostaining for Glut-1 (P < .001). Overall, Glut-1 expression was significantly associated with high histologic grade, ER negativity, PR negativity, CK5/6 positivity, EGFR expression, and high p53 expression (P < .001). However, there was no correlation between Glut-1 immunostaining and patient's outcome. CONCLUSIONS: Our results show that Glut-1 is significantly associated with BLBC and might be a potential therapeutic target for this aggressive subgroup of breast cancer, and this warrants further investigations.

Translational Oncology (2011) 4, 321-327

Translational Oncology

www.transonc.com •

Volume 4 Number 6 December 2011

pp. 321-327 321

Glut-1 Expression Correlates with Basal-like Breast Cancer1

Yaser R. Hussein*, Sudeshna Bandyopadhyay*, Assaad Semaanf, Quratulain Ahmed*, Bassam Albashiti*, Tarek Jazaerly*, Zeina Nahleh* and Rouba Ali-Fehmi*

*Department of Pathology, Wayne State University School of Medicine, Detroit, MI, USA; department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA; ^Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI, USA

Introduction

Recent gene expression profiling studies on breast tumors have identified five distinct subtypes of breast cancer (luminal A, luminal B, human epidermal growth factor receptor 2 [HER2] overexpressing, basal-like, and normal-like) with different clinical outcomes [1-3]. Luminal (steroid receptor-positive) tumors have a superior outcome compared with HER2 or basal-like subtypes. Basal-like breast cancer (BLBC) is characterized by constitutive expression of genes usually found in normal basal/myoepithelial cells of the breast [4].

There is no consensus on how to define BLBC by immunohisto-chemical (IHC) markers. The majority of BLBCs lack the expression of estrogen receptor (ER), progesterone receptor (PR), and Her2 protein overexpression [5-7]. BLBC also has been characterized by the expression of basal cytokeratins (CKs) 5/6 and 17, epidermal growth factor

receptor (EGFR), c-kit, and vascular endothelial growth factor (VEGF) [1,7,8]. Nielsen et al. [7] have developed an IHC panel for identifying BLBCs on the basis of a comparison between the transcriptomic and IHC profiles. According to this definition, BLBCs are negative for ER and HER2 and positive for CK5/6 and/or EGFR. Conversely, others have proposed that a proportion of BLBCs may be positive for ER and Her2 [9,10].

Address all correspondence to: Associate Prof. Rouba Ali-Fehmi, MD, Department of Pathology, Wayne State University School of Medicine, 540 E Canfield, Detroit, MI 48201. E-mail: rali@med.wayne.edu 1The authors declare no conflict of interest.

Received 23 August 2011; Revised 23 August 2011; Accepted 23 September 2011

Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1944-7124/11/$25.00 DOI 10.1593/tlo.11256

BLBC has been a particular focus of attention because this pheno-type has no confirmed therapeutic molecular target and has a poor prognosis [4-7]. Identification of new biological key pathways driving BLBC might aid in finding targets of potential interest for therapeutic blockade.

Tumor hypoxia is a key factor driving the development of malignancy. The presence of hypoxia in tumors is known to lead to resistance to radiotherapy and chemotherapy and is associated with a more aggressive phenotype with an increased propensity for metastases [11,12]. This latter characteristic is thought to be related to the increased expression of a number of proteins acting through the hypoxia-inducible factor 1 (HIF-1) pathway, which allows tumor cells to survive the harsh tumor microenvironment. Glucose transporter 1 (Glut-1) is one of the proteins upregulated in hypoxic conditions [13], and its expression is dually controlled through HIF-1 and in response to reduced oxidative phosphorylation [14]. Glut-1 is one of the facilitative cell surface glucose transporter family that function as an energy-independent system for transport of glucose down a concentration gradient [15]. Glut-1 is not detectable in a large proportion of cells from normal tissues except for erythrocytes, germinal cells of the testis, renal tubules, perineurium of peripheral nerves, and endothelial cells in blood-brain barrier vessels [16]. In contrast, overexpression of Glut-1 has been described in various malignant tumors and was associated with enhanced tumor aggressiveness and poor outcome [17-19]. It has been previously demonstrated that Glut-1 expression was increased in poorly differentiated breast carcinoma and associated with high proliferative activity, increased inva-siveness, and aggressive behavior [20-22].

To our knowledge, published data on the expression of Glut-1 in BLBC are scarce. Therefore, the main objective of this study was to characterize the IHC expression of Glut-1 in patients with BLBC compared to non-BLBC using a panel of IHC stains.

Materials and Methods

Patients and Data Collection

We identified 523 cases of invasive breast cancer in the database of our institution diagnosed between 2004 and 2006, for which paraffin blocks were available. After obtaining approval from the institutional review board, a retrospective chart review of patients' demographic, clinical, and pathological data was performed.

Patients who received preoperative treatment were excluded from this study. Tumor histology, tumor grade, lymph node status, stage, ER, PR, and HER2 status were determined from the original pathology reports. Tumors had been diagnosed in our institution by experienced pathologists using standard criteria for histology and modified Scarff-Bloom-Richardson criteria for grade [23]. Based on the histologic subtype, tumors were assigned to one of the following groups: 1) invasive ductal carcinoma not otherwise specified or any other special type of invasive ductal carcinoma, 2) invasive lobular carcinoma, 3) mixed ductal and lobular carcinoma, or 4) adenocarcinoma with spindle cell metaplasia and metaplastic carcinoma.

Tumors were considered to be positive for ER or PR when nuclear reactivity was observed in at least 1% of neoplastic cells with an intensity of 3+ [24]. The expression of Her2 was classified according to the Hercept Test assay's scoring system, which includes four categories, namely, 0, 1+, 2+, and 3+, based on the intensity and proportion of membrane staining in tumor cells. Positivity was defined as a Her2 score of 3+ for immunostaining (>30% of the tumor cells show circumferential intense and uniform staining) or a >2.2-fold increase

in Her2 gene amplification, as determined by fluorescence in situ hybridization using the Vysis PathVysion Her-2 DNA Probe Kit (Abbott Molecular, Inc, Abbott Park, IL) [25]. ER, PR, and Her2 tests were done at the time of initial diagnosis on needle core biopsies or excision/mastectomy specimens, with a minimum 6-hour fixation time in formaldehyde. The methodology and cutoffs for ER, PR, and Her2 were the same for all the cases included in this study. Stage of the tumor at diagnosis was assigned according to the American Joint Committee on Cancer [26]. Follow-up for patients was obtained from our Computer Information System records and the Surveillance Epidemiology and End Results database. The overall survival was the time, in months, from the date of the primary surgery to the time of breast cancer-related death. The median and mean follow-up time was 41.1 months (range, 0-72 months) and 40.2 months, respectively.

Tissue Samples

The hematoxylin and eosin slides of all the cases were reviewed, and appropriate areas from the tumors were selected for tissue microarray construction (TMAs). TMAs were prepared using selected paraffin-embedded blocks of tumor from each case. Two 1-mm cores were obtained from each block. A total of 19 TMAs were performed. Each TMA consists of 62 cores containing 2 cores from each patient and normal tonsil and ovary as controls. This procedure has been validated in previous breast cancer studies [27].

IHC Techniques

IHC stains including CK5/6, EGFR, p53, and Glut-1 were performed on TMA sections. Five-micrometer-thick unstained sections were placed onto glass slides, then deparaffinized in xylene, and rehy-drated through a series of decreasing ethanol concentration. The sections were pretreated with hydrogen peroxide (3%) for 10 minutes to remove the endogenous peroxidase, followed by antigen retrieval through steam bath for 20 minutes in citrate buffer. The primary antibody was applied, followed by washing and incubation with the biotiny-lated secondary antibody for 30 minutes at room temperature. Antigen detection was carried out by placing diaminobenzidine on each section. The slides were counterstained with hematoxylin and dehydrated in alcohol and xylene before the slides were mounted. The characteristics of the primary antibodies used in this study are listed in Table 1.

Evaluation of IHC Staining

The expression of CK5/6 and EGFR was designated as positive if any cytoplasmic and/or membranous staining was observed [4]. Positivity for Glut-1 was defined as any detectable membranous staining in tumor cells. In contrast, cases designated as -1-negative did not reveal any IHC staining for Glut-1 [28,29]. The cutoff point between

Table 1. Characteristics of the Primary Antibodies.

Marker Clone Species Manufacturer* Dilution (Duration) Antigen Retrieval

ER ER-6F11 Mouse mAb Dako Predilute (32 min) Citrate buffer

PR PGR636 Mouse mAb Dako Predilute (32 min) Citrate buffer

Her2 CB11 Mouse mAb Dako Predilute (32 min) Citrate buffer

CK5/6 D5/16B4 Mouse mAb Cell Marque Predilute (32 min) Citrate buffer

EGFR 31G7 Mouse mAb Ventana Predilute (32 min) Citrate buffer

p53 DO-7 Mouse mAb Ventana Predilute (32 min) Citrate buffer

Glut-1 E308 Rabbit poly Dako 1:100 (1 h) Citrate buffer

mAb indicates monoclonal; poly, polyclonal.

*Dako, Carpinteria, CA; Cell Marque, Rocklin, CA; Ventana, Tucson, AZ.

Figure 1. Photomicrograph of a BLBC showing a high-grade invasive ductal carcinoma not otherwise specified, composed of broad sheets of polygonal tumor cells, with marked pleomorphism and areas of necrosis. (A) Staining with hematoxylin-eosin stain (magnification, x100). Strong immunoreactivity of cytokeratin5/6 (B) and EGFR (C) in tumor cells (magnification, x200).

low and high p53 expression was 50% [30]. A positive control with a tissue sample known to express the antigen of interest was included with each stain. Red blood cells within tissue sections from a capillary hemangioma case were used as positive controls for Glut-1. Three pathologists (Y.H., R.A., and S.B.) individually evaluated the slides blindly under a transmission light microscope. The concordance rate was 90% between the three pathologists. In case of disagreement, the slides were reviewed simultaneously by the three pathologists seated at a multiheaded microscope with a resolution of the difference in opinion.

Definition of BLBC

There is no consensus on how to define BLBC based on immuno-histochemistry. Most of the BLBCs lack the expression of ER, PR, and Her2 and express one of the basal cytokeratins like CK5/6 or CK17 [5-7]. Nielsen et al. [7] developed a classification based on the lack ofER, PR, and Her2 expression, coupled with the expression ofCK5/6 and or EGFR. This panel identified gene expression-based BLBC with a sensitivity of 76% and a specificity of 100%. Several studies indicate that BLBC can be reliably defined by the absence of ER, PR, and Her2 expression (i.e., triple-negative) and therefore triple-negative and BLBC should be synonymous. Conversely, others have proposed that a proportion of BLBCs may be positive for ER and Her2 [9,10].

In this study, we defined BLBC using the criteria of Carey et al. and others by the negativity to ER, PR, and Her2 plus the expression CK5/6 and/or EGFR [7,31,32] (Figure 1).

Statistical Analysis

Statistical analysis was performed using SPSS version 17.0 (Chicago, IL). x2 and Fisher exact tests were used to study the statistical association between clinicopathologic and IHC variables. Unpaired t test was used for analysis of continuous variables. Survival times were estimated in months from the date of diagnosis to the date of death or last follow-up. Survival curves were plotted using the Kaplan-Meier method, and differences in survival curves were assessed by the log-rank test. Statistical significance was defined as a P < .05.

Results

We identified 523 cases of invasive breast carcinoma in the database of our institution for which paraffin blocks and ER, PR, and Her2 markers were available. The mean age of patients was 56.9 years (range, 26-94 years). The median follow-up time was 41.1 months

(range, 0-72 months). The clinicopathologic characteristics of all the patients enrolled in this study are summarized in Table 2.

There was 20% loss of cases either due to loss of tissue cores during processing or due to lack of tumor cells. The interpretable cases were still representative of the total cases (423/523 cases) in various clinicopathologic variables including age, tumor grade, stage, and hormonal status.

The correlation of Glut-1 expression with various clinicopathologic features and biologic markers is detailed in Table 3. There was a significant difference in the mean age at diagnosis between Glut-1-positive

Table 2. Clinicopathologic Characteristics in 523 Invasive Breast Carcinomas.

Age, mean ± SD, years 56.9 ± 11.7

Race, n (%)

African American 317 (60.6)

White 196 (37.5)

Others 10 (1.9)

Histologic grade, n (%)

1 32 (6.1)

2 128 (24.5)

3 311 (59.5)

Unknown 52 (9.9)

Histologic subtype,* n (%)

IDC 456 (87.9)

ILC 39 (7.5)

Mixed 18 (3.4)

Others 6 (1.1)

Tumor size, n (%), cm

<2 178 (34)

2-5 135 (25.8)

5-10 45 (8.6)

>10 18 (3.4)

Unknown 147 (28.2)

Stage, n (%)

I 128 (24.5)

II 177 (33.9)

III 92 (17.6)

IV 40 (7.6)

Unknown 86 (16.4)

ER, n (%)

Negative 203 (38.8)

Positive 320 (61.2)

PR, n (%)

Negative 156 (29.8)

Positive 367 (70.2)

Her2, n (%)

Negative 463 (88.5)

Positive 60 (11.5)

IDC indicates invasive ductal carcinoma; ILC, invasive *Data are lacking for some patients. lobular carcinoma.

Table 3. Correlation of Glut-1 Expression with Clinicopathologic Characteristics and Biologic Markers.

Variable No. Patients Glut-1 Negative (n = 214, 65%) Glut-1 Positive (n = 115, 35%) P

Age, mean ± SD, years 329 58.8 ± 14.1 55.1 ± 13.4 .02

Race, n (%)

African American 193 116 (60.1) 77 (39.9) .07

White 129 93 (72.1) 36 (27.9)

Others 4 3 (75) 1 (25)

Histologic grade,* n (%)

1 25 19 (76) 6 (24) .01

2 81 61 (75.3) 20 (24.7)

3 191 108 (56.5) 83 (43.5)

Histologic subtype, n (%)

IDC 284 180 (63.4) 104 (36.6) <.001

ILC 26 25 (96.2) 1 (3.8)

Mixed 13 9 (69.2) 4 (30.8)

Others 6 0 6 (100)

Tumor size, n (%), cm

<2 130 77 (59.2) 53 (40.8) .14

2-5 60 39 (65) 21 (35)

5-10 22 17 (77.3) 5 (22.7)

>10 9 8 (88.9) 1 (11.1)

Lymph node,* n (%)

Negative 131 78 (59.5) 53 (40.5) .09

Positive 114 79 (69.3) 35 (30.7)

American Joint Committee on Cancer stage,* n (%)

1 76 53 (69.7) 23 (30.3) .35

2 98 57 (58.2) 41 (41.8)

3 41 29 (70.7) 12 (29.3)

4 8 5 (62.5) 3 (37.5)

ER, n (%)

Negative 102 40 (39.2) 62 (60.8) <.001

Positive 227 174 (76.7) 53 (23.3)

PR, n (%)

Negative 209 120 (57.4) 89 (42.6) <.001

Positive 120 94 (78.3) 26 (21.7)

Her2, n (%)

Negative 302 198 (65.6) 104 (34.4) .53

Positive 27 16 (59.3) 11 (40.7)

CK5/6,f n (%)

Negative 186 140 (75.3) 46 (24.7) <.001

Positive 126 63 (50) 63 (50)

EGFR,f n (%)

Negative 210 159 (75.7) 51 (24.3) <.001

Positive 92 39 (42.4) 53 (57.6)

p53,f n (%)

Low (<50%) 236 166 (70.3) 70 (29.7) <.001

High (>50%) 69 30 (43.5) 39 (56.5)

*Data are lacking for some patients.

^Some tissue cores are missing due to processing.

and Glut-1-negative cases (mean age, 58.8 vs 55.1 years, respectively, P = .02). Glut-1 positivity was significantly associated with invasive ductal carcinoma, compared to invasive lobular carcinoma and mixed ductal and lobular carcinoma (P < .001). Although the association between Glut-1 expression and axillary lymph node status at diagnosis was not significant (P = .09), tumors positive for Glut-1 were more likely to be of negative lymph node status.

Glut-1 expression was significantly associated with high histologic grade, ER negativity, PR negativity, CK5/6 positivity, EGFR expression, and high p53 expression (P < .001). There was no significant association between Glut-1 expression and race, tumor size, pathologic stage, and Her2 status.

When we defined BLBC by negativity to ER, PR, and Her2 plus the expression CK5/6 and/or EGFR), 62 cases (14.7%) were categorized as BLBC versus 361 (85.3%) non-BLBC (Figure 1). In this study, we found a significant correlation between Glut-1 expression and BLBC (Table 4). Glut-1 was expressed in 42 (76.4%) of 55 BLBCs, whereas only 55 (23.8%) of 231 non-BLBCs showed expression for

Glut-1 (P < .001). Figure 2 shows a case of BLBC with positive Glut-1 expression and a case of non-BLBC with negative Glut-1 staining. When we stratified both categories by grade, Glut-1 was still significantly associated with BLBC. Among the poorly differentiated tumors, Glut-1 was expressed in 71% of BLBCs versus 29% of non-BLBCs (P < .001).

Glut-1 expression was associated with lower overall survival rate; however, it did not reach significance. At 5 years, the overall survival rate was 65.8% for patients with Glut-1-positive tumors versus 73.8% for patients with Glut-1-negative tumors (P = .13; Figure 3). Similarly,

Table 4. Correlation of Glut-1 Expression with the Molecular Subtypes of Breast Cancer.

Glut-1, n (%) BLBC (n = 55, 19.5%) Non-BLBC (n = 231, 80.5%)

Positive 42 (76.4) 55 (23.8)

Negative 13 (23.6) 176 (76.2)

P <.001

Figure 2. (A) BLBC (invasive ductal carcinoma not otherwise specified) showing strong membranous staining for Glut-1 in tumor cells (magnification, x400). (B) Non-BLBC showing tumor cells with negative staining for Glut-1 (magnification, x400).

when we stratified BLBCs according to Glut-1 expression, there was no significant difference in overall survival between the two groups (P = .08).

Discussion

The aim of this study was to compare the IHC expression of Glut-1 between BLBC and non-BLBC using a restricted panel of IHC stains. In this report, we found significant correlation between Glut-1 expression and BLBC.

Basal increased glucose uptake and use is a major feature of malignant tumors compared to normal tissue [33]. This uptake is mediated by glucose transporters and regulated by endogens and growth factors [34]. Glut-1 is one of the facilitative cell surface glucose transporter family that function as an energy-independent system for transport of glucose down a concentration gradient [11] and is dually controlled through HIF-1 and in response to reduced oxidative phosphorylation [14]. Overexpression of Glut-1 has been described in various malignancies such as non-small cell lung carcinoma, colorectal carcinoma, and gastric carcinoma; and it was associated with enhanced tumor aggressiveness and poor outcome [17-19].

Previous studies on the expression of Glut-1 in breast cancer showed that Glut-1 expression was associated with high nuclear grade, high proliferative activity, and aggressive behavior [20-22]. We found that Glut-1 expression was significantly associated with high histologic grade, ER and PR negativity, and basal cytokeratins expression.

Younes et al. [22] found positive correlation between Glut-1 expression and nuclear grade. In contrast to our results, Glut-1 immunostaining did not correlate with ER status. This contradictory result regarding ER status could be explained due to the use of different techniques. They assessed ER status using the dextran charcoal assay with sucrose gradient centrifugation method on frozen breast tissue. Kang et al. [35] demonstrated that Glut-1 expression correlated significantly with high nuclear grade, ER and PR negativity, overall survival, and disease-free survival. In our study, Glut-1 expression was associated with lower overall survival rate; however, it did not reach significance. This could be explained by the relatively short follow-up time ofour patients. Similar to our results, both studies found that Glut-1 expression did not correlate with tumor size and lymph node involvement.

There was a significant association between Glut-1 immunostaining and high p53 expression, which is considered as a biological marker of aggressiveness. It has been shown that the wild-type p53 represses Glut-1 gene transcription; in contrast, the mutated p53 is associated with up-regulation of the transcriptional activity of the Glut-1 gene promoters. This, in turn, results in increased glucose metabolism and cell energy supply, which would be predicted to facilitate tumor growth [36].

We found significant correlation between Glut-1 expression and BLBC. BLBC has been characterized by expression of EGFR, c-kit, VEGF, and higher incidence of p53 and breast cancer 1 (BRCA1) mutations [4-7].

van der Groep et al. investigated the association between HIF-1 and Glut-1 with BRCA1 germline mutation-related breast cancer [37]. BRCA1 related breast cancers are often of high-grade, high-proliferative activity, triple-negative phenotype and often express basal cytokeratins (i.e., BLBC) [38-40]. Overexpression of Glut-1 and HIF-1 was significantly more frequent in BRCA1 germline mutation-related cancer, that is, 27 of30 (90%) versus 54 (29%) of 183 and 88 (44%) of200 sporadic control breast cancer cases [37].

Because chemotherapy is the only therapeutic option currently available for patients with BLBC, research during recent years has focused on increasing knowledge about key pathways in BLBC aimed at discovery of new treatment options. Novel targeted therapies (e.g., EGFR, c-kit, and VEGF inhibitors alone or in combination with chemotherapy) have been investigated with mixed results [41-43].

Hypoxic pathways might be a potential target for this breast cancer subtype. Pharmacological inhibition of glucose metabolism has been shown to exhibit promising anticancer activity in vitro and in vivo, alone or in combination with other therapeutic modalities. One study showed that anti-Glut-1 antibodies inhibit proliferation and

Figure 3. Kaplan-Meier short-term overall survival by Glut-1 expression.

induce apoptosis in breast cancer cell lines. Furthermore, it showed that anti-Glut-1 antibodies enhance apoptosis caused by chemo-therapeutic agents like cisplatin, paclitaxel, and targeted agents like gefitinib [44]. Glufosfamide is a cytotoxic agent delivered through the glucose transport system. In a feasibility trial of this agent, complete response was observed in pancreatic, colon, and breast cancer patients [45]. Inhibition of expression or functionality of Glut-1, rather than inhibiting glucose metabolism in its entirety may more specifically target those cells within the tumor that depend on a high rate of glucose uptake and glycolysis [46].

The current study has its limitations. These include 1) loss of some cases either due to loss of tissue cores during processing or due to lack of tumor cells, 2) the relatively short follow-up time of our patients, and 3) the fact that the use of IHC to evaluate protein levels does not always reflect the structure or functionality of the protein. However, the strength of our study lies in the fact that, to our knowledge, the current study is the first to demonstrate an association between Glut-1 expression and BLBC.

In conclusion, our results show that Glut-1 is significantly correlated with BLBC and might be a potential therapeutic target for this aggressive subtype of breast cancer, and this warrants further investigation.

References

[1] Brenton JD, Carey LA, Ahmed AA, and Caldas C (2005). Molecular classification and molecular forecasting of breast cancer: ready for clinical application? J Clin Oncol 23(29), 7350-7360.

[2] Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, et al. (2000). Molecular portraits of human breast tumours. Nature 406(6797), 747-752.

[3] Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, et al. (2003). Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100(14), 8418-8423.

[4] S0rlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, et al. (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98(19), 10869-10874.

[5] Abd El-Rehim DM, Ball G, Pinder SE, Rakha E, Paish C, Robertson JF, Macmillan D, Blamey RW, and Ellis IO (2005). High-throughput protein expression analysis using tissue microarray technology of a large well-characterised series identifies biologically distinct classes of breast cancer confirming recent cDNA expression analyses. Int J Cancer 116(3), 340-350.

[6] Abd El-Rehim DM, Pinder SE, Paish CE, Bell J, Blamey RW, Robertson JF, Nicholson RI, and Ellis IO (2004). Expression of luminal and basal cytokeratins in human breast carcinoma./Pathol 203(2), 661-671.

[7] Nielsen TO, Hsu FD, Jensen K, Cheang M, Karaca G, Hu Z, Hernandez-Boussard T, Livasy C, Cowan D, Dressler L, et al. (2004). Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10(16), 5367-5374.

[8] Linderholm BK, Hellborg H, Johansson U, Elmberger G, Skoog L, Lehtio J, and Lewensohn R (2009). Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer. Ann Oncol 20(10), 1639-1646.

[9] Fulford LG, Easton DF, Reis-Filho JS, Sofronis A, Gillett CE, Lakhani SR, and Hanby A (2006). Specific morphological features predictive for the basal phenotype in grade 3 invasive ductal carcinoma of breast. Histopathology 49(1), 22-34.

[10] Matos I, Dufloth R, Alvarenga M, Zeferino LC, and Schmitt F (2005). p63, cytokeratin 5, and P-cadherin: three molecular markers to distinguish basal phenotype in breast carcinomas. Virchows Arch 447(4), 688-694.

[11] Brown JM and Giaccia AJ (1998). The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 58(7), 1408-1416.

[12] Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, and Vaupel P (1996). Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 56(19), 4509-4515.

[13] Semenza GL (2000). Hypoxia, clonal selection, and the role of HIF-1 in tumor progression. Crit Rev Biochem Mot Biol 35(2), 71—103.

[14] Behrooz A and Ismail-Beigi F (1997). Dual control of glut1 glucose transporter gene expression by hypoxia and by inhibition of oxidative phosphorylation. J Biol Chem 272(9), 5555-5562.

[15] Olson AL and Pessin JE (1996). Structure, function, and regulation of the mammalian facilitative glucose transporter gene family. Annu Rev Nutr 16,

235-256.

[16] Godoy A, Ulloa V, Rodriguez F, Reinicke K, Yanez AJ, Garcia Mde L, Medina RA, Carrasco M, Barberis S, Castro T, et al. (2006). Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues. J Cell Physiol 207(3), 614-627.

[17] Haber RS, Rathan A, Weiser KR, Pritsker A, Itzkowitz SH, Bodian C, Slater G, Weiss A, and Burstein DE (1998). GLUT1 glucose transporter expression in colorectal carcinoma: a marker for poor prognosis. Cancer 83(1), 34-40.

[18] Kawamura T, Kusakabe T, Sugino T, Watanabe K, Fukuda T, Nashimoto A, Honma K, and Suzuki T (2001). Expression of glucose transporter-1 in human gastric carcinoma: association with tumor aggressiveness, metastasis, and patient survival. Cancer 92(3), 634-641.

[19] Younes M, Brown RW, Stephenson M, Gondo M, and Cagle PT (1997). Overexpression of Glut1 and Glut3 in stage I nonsmall cell lung carcinoma is associated with poor survival. Cancer 80(6), 1046-1051.

[20] Alo PL, Visca P, Botti C, Galati GM, Sebastiani V, Andreano T, Di Tondo U, and Pizer ES (2001). Immunohistochemical expression of human erythrocyte glucose transporter and fatty acid synthase in infiltrating breast carcinomas and adjacent typical/atypical hyperplastic or normal breast tissue. Am J Clin Pathol 116(1), 129-134.

[21] Grover-McKay M, Walsh SA, Seftor EA, Thomas PA, and Hendrix MJ (1998). Role for glucose transporter 1 protein in human breast cancer. Pathol Oncol Res 4(2), 115-120.

[22] Younes M, Brown RW, Mody DR, Fernandez L, and Laucirica R (1995). GLUT1 expression in human breast carcinoma: correlation with known prognostic markers. Anticancer Res 15(6B), 2895-2898.

[23] Elston CWand Ellis IO (1991). Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 19(5), 403-410.

[24] Fitzgibbons PL, Murphy DA, Hammond ME, Allred DC, and Valenstein PN (2010). Recommendations for validating estrogen and progesterone receptor immunohistochemistry assays. Arch Pathol Lab Med 134, 930-935.

[25] Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, et al. (2007). American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25(1), 118-145.

[26] Greene FL and American Joint Committee on Cancer (2002). AJCC Cancer Staging Manual (6th ed). Springer, New York, NY. xiv, p. 421.

[27] Camp RL, Charette LA, and Rimm DL (2000). Validation of tissue microarray technology in breast carcinoma. Lab Invest 80(12), 1943-1949.

[28] Amann T, Maegdefrau U, Hartmann A, Agaimy A, Marienhagen J, Weiss TS, Stoeltzing O, Warnecke C, Schölmerich J, Oefner PJ, et al. (2009). GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol 174(4), 1544-1552.

[29] Younes M, Lechago LV, Somoano JR, Mosharaf M, and Lechago J (1996). Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers. Cancer Res 56(5), 1164-1167.

[30] Alkushi A, Lim P, Coldman A, Huntsman D, Miller D, and Gilks CB (2004). Interpretation of p53 immunoreactivity in endometrial carcinoma: establishing a clinically relevant cut-off level. Int J Gynecol Pathol 23(2), 129-137.

[31] Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, et al. (2006). Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295(21), 2492-2502.

[32] Spitale A, Mazzola P, Soldini D, Mazzucchelli L, and Bordoni A (2009). Breast cancer classification according to immunohistochemical markers: clinicopatho-logic features and short-term survival analysis in a population-based study from the South of Switzerland. Ann Oncol 20(4), 628-635.

[33] Isselbacher KJ (1972). Sugar and amino acid transport by cells in culture-differences between normal and malignant cells. NEngl JMed 286(17), 929-933.

[34] Merrall NW, Plevin R, and Gould GW (1993). Growth factors, mitogens, oncogenes and the regulation of glucose transport. Cell Signal 5(6), 667—675.

[35] Kang SS, Chun YK, Hur MH, Lee HK, Kim YJ, Hong SR, Lee JH, Lee SG, and Park YK (2002). Clinical significance of glucose transporter 1 (GLUT1) expression in human breast carcinoma. Jpn J Cancer Res 93(10), 1123—1128.

[36] Schwartzenberg-Bar-Yoseph F, Armoni M, and Karnieli E (2004). The tumor suppressor p53 down-regulates glucose transporters GLUT1 and GLUT4 gene expression. Cancer Res 64(7), 2627—2633.

[37] van der Groep P, Bouter A, Menko FH, van der Wall E, and van Diest PJ (2008). High frequency of HIF-1a overexpression in BRCA1 related breast cancer. Breast Cancer Res Treat 111(3), 475—480.

[38] Chappuis PO, Nethercot V, and Foulkes WD (2000). Clinico-pathological characteristics of BRCA1- and BRCA2-related breast cancer. Semin Surg Oncol 18(4), 287-295.

[39] Eisinger F, Stoppa-Lyonnet D, Longy M, Kerangueven F, Noguchi T, Bailly C, Vincent-Salomon A, Jacquemier J, Birnbaum D, and Sobol H (1996). Germ line mutation at BRCA1 affects the histoprognostic grade in hereditary breast cancer. Cancer Res 56(3), 471 -474.

[40] Foulkes WD, Brunet JS, Stefansson IM, Straume O, Chappuis PO, Bégin LR, Hamel N, Goffin JR, Wong N, Trudel M, et al. (2004). The prognostic implication of the basal-like (cyclin E high/p27 low/p53+/glomeruloid-microvascular-proliferation+) phenotype of BRCA1-related breast cancer. Cancer Res 64(3), 830-835.

[41] Corkery B, Crown J, Clynes M, and O'Donovan N (2009). Epidermal growth factor receptor as a potential therapeutic target in triple-negative breast cancer. Ann Oncol 20(5), 862-867.

[42] Finn RS, Bengala C, Ibrahim N, Strauss LC, Fairchild J, Sy O, Roché H, Sparano J, and Goldstein LJ (2008). Phase II trial of dasatinib in triple-negative breast cancer: results of study CA180059. Cancer Res 69(suppl), 242s. Abstract 3118.

[43] Miller K, Wang M, Gralow J, Dickler M, Cobleigh M, Perez EA, Shenkier T, Cella D, and Davidson NE (2007). Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med 357(26), 2666-2676.

[44] Rastogi S, Banerjee S, Chellappan S, and Simon GR (2007). Glut-1 antibodies induce growth arrest and apoptosis in human cancer cell lines. Cancer Lett 257(2),

244-251.

[45] Briasoulis E, Judson I, Pavlidis N, Beale P, Wanders J, Groot Y, Veerman G, Schuessler M, Niebch G, Siamopoulos K, et al. (2000). Phase I trial of 6-hour infusion of glufosfamide, a new alkylating agent with potentially enhanced selectivity for tumors that overexpress transmembrane 452 glucose transporters: a study of the European Organization for Research and Treatment of Cancer Early Clinical Studies Group. J Clin Oncol 18, 3535-3544.

[46] Evans A, Bates V, Troy H, Hewitt S, Holbeck S, Chung YL, Phillips R, Stubbs M, Griffiths J, and Airley R (2008). Increased chemoresistance and HIF-1 independent link with cell turnover is revealed through COMPARE analysis and metabolic studies. Cancer Chemother Pharmacol 61, 377-393.