Negative Breast Cancer Patients

2344 Vol. 10, 2344–2350, April 1, 2004 Clinical Cancer Research

Prognostic Impact of ANX7-GTPase in Metastatic and HER2- Negative Breast Cancer Patients

Meera Srivastava,1 Lukas Bubendorf,2 of survival decreases by more than 10-fold for those patients Mark Raffeld,3 Christoph Bucher,2 with HER2-negative tumors. These latter patients repre- Jochen Torhorst,2 Guido Sauter,2 Cara Olsen,4 sented 66% of the population affected with breast cancer in this study. Olli P. Kallioniemi,5 Ofer Eidelman,1 and 1 Conclusions: High levels of ANX7 in tumor correlate Harvey B. Pollard strongly with poor survival of HER2-negative patients and 1 Department of Anatomy, Physiology and Genetics and Institute for the most aggressive forms of breast cancer. This is the first Molecular Medicine, Uniformed Services University School of Medicine, Bethesda, Maryland; 2Institute for Pathology, University of study to demonstrate that ANX7 antibody has the potential Basel, Basel, Switzerland; 3Laboratory of Pathology, for development into an in vivo diagnostic and therapeutic Hematopathology Section, National Cancer Institute, NIH, Bethesda, tool. This simple and reliable immunohistochemical assay 4 Maryland; Preventive Medicine and Biometrics, Uniformed Services may therefore become an important biomarker for meta- University School of Medicine, Bethesda, Maryland; and 5Section on Molecular Genetics, Cancer Genetics Branch, National Human static breast cancer diagnosis and management of HER2- Genome Research Institute, NIH, Bethesda, Maryland negative breast tumor patients.

ABSTRACT INTRODUCTION Purpose: ANX7-GTPase located on chromosome 10q21 Breast cancer is the most common malignancy among is significantly altered and associated with hormone-refrac- women in developed countries, and there is considerable need tory metastatic prostate cancers. Therefore, we investigated for reliable prognostic markers to assist clinicians in making whether levels of ANX7 correlate with breast cancer pro- diagnostic and therapeutic management decisions. A number of gression and survival biological factors have been used to define risk categories in Experimental Design: A diagnostic tumor tissue mi- breast cancer. We have focused on the role of ANX7-GTPase in croarray containing 525 human breast tissue specimens at breast cancer progression and survival because altered expres- different stages of the disease was assayed for ANX7 using sion of this gene is associated with metastatic and hormone- immunocytochemical methods with ANX7 monoclonal anti- refractory prostate cancer in man and with a tumor-prone phe- body. A separate prognostic tumor tissue microarray con- notype in the Anx7(ϩ/Ϫ) mouse model (1–3). The ANX7 gene taining 553 human breast tissue specimens annotated with product is a Ca2ϩ-activated GTPase (4) and protein kinase C clinicopathological parameters was assayed for ANX7, substrate (5, 6), which shares with other members of the annexin HER2, estrogen receptor, progesterone receptor, and p53 gene family the ability to bind to acidic phospholipids in a protein. Ca2ϩ-dependent manner (7). ANX7 also forms classical volt- Results: We report here for the first time that the age-gated Ca2ϩ channels in cellular and artificial membranes expression of ANX7-GTPase is significantly enhanced and (8). The role of calcium in many important cell processes is well associated with the presence of metastatic disease (P < appreciated, although the involvement of calcium-binding pro- 0.0001) in the 525 human breast tissue specimens analyzed. teins in the annexin gene family for cancer remains somewhat Furthermore, using a separate 553 case retrospective prog- novel (2, 3, 9–11). nostic tumor tissue microarray, we found that increased Recent studies from our laboratory indicate that altered ANX7 expression is also significantly associated with poor expression of ANX7 is associated with metastatic and hormone- overall patient survival (P < 0.014). This is particularly true refractory prostate cancer. We have therefore hypothesized that when restricted to patients in whom the BRE clinical grade ANX7 signaling might play a fundamental role in breast cancer is2(P 0.001) or for whom there is a lack of HER2 < occurrence and progression. To test this hypothesis, we have expression (P 0.002). Finally, Cox regression analysis < used breast tissue microarrays containing approximately 1078 shows that as the expression of ANX7 rises, the probability biopsy specimens to ask whether the levels of expression of ANX7 might have predictive value for diagnosis and survival of these patients. We report here that increased ANX7 expression is associated with the presence of metastatic disease. As the Received 9/24/03; revised 12/23/03; accepted 1/2/04. expression of ANX7 rises, the probability of survival decreases The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked by more than 10-fold for those patients with tumors that lack advertisement in accordance with 18 U.S.C. Section 1734 solely to HER2 immunostaining. The latter population represents 66% of indicate this fact. breast cancer-affected patients in this series. These findings Requests for reprints: Meera Srivastava, Department of Anatomy, suggest that overexpression of ANX7 can be used as a risk Physiology and Genetics, Uniformed Services University School of Medicine, 4301 Jones Bridge Road, Bethesda. MD 20814. Phone: biomarker that may prove to be useful in making aggressive (301) 295-3204; Fax: (301) 295-1786; E-mail: [email protected]. treatment decisions for breast cancer patients

MATERIALS AND METHODS DAKO), HER2 (Hercep test; DAKO) p53 (DO-7; prediluted; Study Population. The first set of diagnostic progression DAKO), estrogen receptor [ER (ER ID5; 1:1000; DAKO)], and breast cancer tissue microarray contained samples from 525 progesterone receptor [PR (NCL-PGR; 1A6; 1:600; Novocastra breast tissues. The patient group consisted of 107 patients with Laboratories Ltd., Newcastle upon Tyne, United Kingdom; Ref. primary breast cancers, 23 patients with ductal carcinoma in 13)]. Tumors with known positivity were used as positive con- situ, 343 patients with metastatic invasive ductal carcinoma, and trols. The primary antibody was omitted for negative controls. 52 patients with metastatic invasive lobular carcinoma. The These arrays have previously been tested for lack of interaction second prognostic breast tissue microarray contained carcino- with irrelevant monoclonal antibodies. Scoring of the immuno- mas of 553 breast cancer patients for whom follow-up data histochemical staining followed the guidelines in the package ϫ (tumor-specific survival and treatment information) could be insert using an objective at 10 magnification. The ANX7 levels were classified as 0 (no staining), 1 (low staining), 2 evaluated retrospectively. These patients had a median age of 61 (moderate staining), and 3 (highest staining intensity). Tumors years (range, 33–97 years). They were treated for primary breast were considered positive for ANX7 if an unequivocal nuclear or cancer at the University Hospital of Basel (Basel, Switzerland), cytoplasmic positivity was seen in at least 10% of tumor cells. Women’s Hospital (Rheinfelden, Germany), and the Kreiskran- Immunohistochemical scoring of p53, ER, and PR was done as kenhaus (Lo¨rrach, Germany) between 1985 and 1994. The me- described previously (13). The ANX7 monoclonal antibody has dian potential follow-up time was 63.0 months (range, 1–151 been shown to recognize ANX7 specifically and has proved to months). Formalin-fixed, paraffin-embedded tumor material be a useful reagent for immunohistochemical studies (2). The from both arrays was available from the Institute of Pathology, staining is both nuclear and cytoplasmic, as expected for a University of Basel. The pathological stage, tumor diameter, and protein localized to the nucleus and cytoplasm. The specificity nodal status were obtained from the primary pathology reports. of tissue staining was determined by the demonstration of neg- All slides from all tumors were reviewed by one pathologist ative staining either by omitting primary antibody or by using an (J. T.) to define the histological grade according to Elston and irrelevant antibody. Ellis (BRE). A systemic therapy after surgery had been per- Western Blotting. The nonmetastatic cell line B231lys formed for 273 patients represented on the prognostic tissue and the metastatic cell line B435lys were obtained from Amer- microarray (TMA), including 172 patients with hormonal therapy alone, 52 patients with cytotoxic therapy alone, and 49 patients with both hormonal and cytotoxic treatment. The progression TMA included 405 ductal cancers, 77 lobular cancers, 17 medullary cancers, 14 mucinous cancers, 11 cribriform cancers, 11 tubular cancers, 7 papillary cancers, 4 apocrine cancers, 3 clear cell cancers, 1 metaplastic cancer, 1 atypical medullar cancer, 1 large cell cancer, 1 small cell cancer, and 1 neuroen- docrine cancer. Among 553 tumors, 27.8% were grade 1, 42.9% were grade 2, and 29.3% were grade 3. The pT stage was pT1 in

39.5% of patients, pT2 in 46.3% of patients, pT3 in 4.9% of patients, and pT4 in 9.3% of patients. The stage could not be determined unequivocally from the pathology reports in six tumors. Axillary lymph nodes had been examined in 519 patients (52.4% pN0, 39.3% pN1, and 8.3% pN2). Stage, grade, and nodal status were strongly associated with tumor-specific survival of our patients (P Ͻ 0.0001 each). Immunohistochemistry. Tumor samples were arrayed as described previously (12). Briefly, H&E-stained sections were made from each selected primary tumor block (donor blocks) to define representative tumor regions. Tissue cylinders with a diameter of 0.6 mm were then punched from each donor block using a custom-made precision instrument (Beecher In- struments, Silver Spring, MD) and brought into a recipient paraffin block eventually containing either 525 or 553 individual samples. Four-␮m sections of the recipient blocks were then cut using an adhesive-coated slide system (Instrumedics Inc.) supporting the cohesion of the 0.6-mm array elements on glass. One section from each of the four replica arrays was used for Fig. 1 Disease progression relative to immunological ANX7 expres- immunohistochemical analysis, as described previously (13). sion (525 patients). The percentage of ANX7-positive samples is plotted The guidelines from the package insert were followed for against different stages of breast cancer starting from primary breast cancer (107 specimens), ductal carcinoma in situ (DCIS; 23 specimens), each antibody. Standard indirect immunoperoxidase procedures metastatic ductal invasive carcinoma (343 specimens), and metastatic (ABC-Elite; Vector Laboratories) in combination with mono- lobular invasive carcinoma (52 specimens). ANX7 positivity increases clonal antibodies were used for detection of ANX7 (1:1000; with disease progression.

Fig. 2 Immunohistochemistry on tumor tissue microarray. Analysis of ANX7 protein in representative clinical specimens of metastatic (A) and nonmetastatic (B) breast carcinomas (ϫ100). Intense cytoplasmic staining is observed in metastatic specimens compared with very weak staining in nonmetastatic specimens.

described by Caohuy and Pollard (5, 6). Briefly, the blot was blocked in a solution of milk (5% milk in PBS with 1% BSA) for 1 h. After overnight exposure to the ANX7 primary monoclonal antibody (1:1000; DAKO), the blot was washed four times in PBS/Tween 20 (0.1%; Sigma). The blot was exposed to

Fig. 3 Western blot analysis of metastatic (B435lys) and nonmetastatic (B231lys) breast tumor cell lines. The cell extract was resolved by 10% SDS-PAGE as described in “Materials and Methods.” ANX7 expression is visualized using monoclonal anti-ANX7 antibody. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used as a control and probed with anti-glyceraldehyde-3-phosphate dehydrogenase.

ican Type Culture Collection and grown according to the man- ufacturer’s instructions. For protein extraction, cells were lysed in buffer consisting of 0.5% 2 M Tris, 3% 5 M NaCl, 1% 500 mM EDTA, 1% Triton, 10% glycerol, and 2 mM vanadate. Cells were left on ice for 5 min to allow cell lysis to reach completion, Fig. 4 Kaplan-Meier survival curve for patients subdivided on the at which point the released material was spun down to remove basis of ANX7 expression (553 patients). The patients whose tumors had very high ANX7 expression had significantly shorter survival than cell debris (5 min at 13,000 rpm). The supernatant was separated patients whose tumors had very weak ANX7 expression (P ϭ 0.014). through a 10% SDS gel. Proteins were then transferred to The 5-year survival is 65% for group 3, 76% for groups 2 and 1, and nitrocellulose paper. Western blot analysis was performed as 95% for group 0.

Table 1 Univariate analysis in terms of clinicopathological cytoplasmic ANX7 expression is systematically increased in risk factors patients with metastatic disease (Fig. 1). For example, in pri- Sample Range or mary breast cancers, the proportion of ANX7 positives is 20%. Variable size category Pa However, the fraction of tumors with increased ANX7 expres- BRE 553 1–3 Ͻ0.001 sion is 60% and 80%, respectively, for lymph node metastases pT 547 1–4 Ͻ0.001 associated with invasive ductal and lobular metastatic breast pN 519 0–2 Ͻ0.001 cancers. Metastases differ from primary carcinomas in a statis- Ͻ HER2 546 0–3 0.001 tically significant manner (P Ͻ 0.0001) using the ␹2 test. Im- ERb 553 Pos. vs. neg. Ͻ0.001 PR 553 Pos. vs. neg. Ͻ0.001 munohistochemical analysis of breast tumor tissue arrays re- p53 553 Pos. vs. neg. 0.066 ANX7 383 0–3 0.014 ANX7, where BRE ϭ 1 103 0–3 0.439 ANX7, where BRE ϭ 2 160 0–3 0.002 ANX7, where BRE ϭ 3 120 0–3 0.892 ANX7, where HER2 ϭ 0 283 0–3 0.002 ANX7, where HER2 Ͼ 0 100 0–3 0.970 a Unadjusted log-rank P calculated by Kaplan-Meier survival analysis. Sample size excludes cases with missing values. b ER, estrogen receptor; PR, progesterone receptor; Pos., positive; neg., negative.

the secondary antibody for 30 min, followed by three washes in the PBS/Tween 20 solution. The blot was soaked in SuperSignal solution (Pierce), dried briefly, wrapped in Saran Wrap, and then exposed to Kodak film for different times at room temper- ature. Statistical Analysis. Initially, actuarial analyses were performed using the Kaplan-Meier method to construct survival curves, which were compared with a log-rank test. Survival time was measured in months from date of surgery until date of death or last follow-up. The following variables were considered (levels compared are in parentheses): BRE (1 versus 2 versus 3); pT (1 versus 2 versus 3 versus 4); pN (0 versus 1 versus 2); ANX7 (0 versus 1 versus 2 versus 3); ER (positive versus negative); PR (positive versus negative); p53 (positive versus negative); HER2 (0 versus 1 versus 2 versus 3); hormone therapy (yes versus no); chemotherapy (yes versus no); neoad- juvant chemotherapy (yes versus no); diameter (divided into four groups based on the quartiles 17, 22, and 30); lymph node positive (x ϭ 0 versus 0 Ͻ x Ͻ 3 versus x Ͼ 3); lymph node all (divided into four groups based on the quartiles 12, 16, and 21); and age (divided into four groups based on the quartiles 51, 61, and 70 years). Survival time was measured in months from date of surgery. Additionally, actuarial analyses were performed on ANX7 by BRE (1 versus 2 versus 3) and HER2 (0 versus 1, 2, or 3). The Cox proportional hazards model method was used to identify the significance of parameters when considered jointly. In addition, a likelihood ratio test was used to determine whether ANX7 was significantly associated with survival after adjust- ment for other common parameters evaluated.

Fig. 5 Survival curve for patients subdivided on the basis of HER2 and RESULTS BRE-2 status. A, at the 5-year survival point, 100% of patients with no High Cytoplasmic ANX7 Expression Is Associated with or very low ANX7 expression survived, whereas only 52% of patients Metastatic Phenotype. To investigate whether there is a re- with strong ANX7 expression survived, and 80% of patients with weak lationship between ANX7 expression and disease progression in to moderate ANX7 expression survived (P ϭ 0.001). B, survival curve for patients subdivided on the basis of ANX7 expression and HER2 is patients with breast cancer, we tested 525 breast specimens from negative. At the 5-year survival point, very low ANX7 expression human primary breast cancers and axillary lymph node metas- predicts 100% survival, whereas high ANX7 expression predicts only tases as well as normal human breast tissues. We found that 60% survival (P ϭ 0.002).

veals strong ANX7 staining in the majority of tumor cells in each of these patient specimens was correlated with patient metastatic tumor specimens, whereas very low ANX7 staining is survival parameters. Four types of ANX7 expression can be observed in nonmetastatic tumors. Representative sections of discriminated in breast cancer specimens. These groups are metastatic and nonmetastatic tissue microarray sections are designated “0” for negative or very low ANX7 expression, “1” shown in Fig. 2, A and B. for weak ANX7 expression, “2” for moderate ANX7 expres- To test whether the high expression levels of ANX7 in sion, and “3” for strong ANX7 expression. As shown in Fig. 4, metastatic cells could be generalized to in vitro conditions, we Kaplan-Meier curves of univariate cumulative survival in pa- examined ANX7 protein expression in relevant cell lines. As tients with low (group 0) versus high (group 3) cytoplasmic shown in Fig. 3, ANX7 levels are very low when assayed by ANX7 expression show a significant separation within 5 years Western analysis in the asynchronously growing human non- of follow-up. The 5-year survival is 65% for group 3 (strong metastatic breast cancer cell line B231lys. In contrast, in the ANX7 expression) and 76% for groups 2 and 1 (moderate metastatic cell line B435lys, strong cytoplasmic staining corre- ANX7 expression). For group 0 (negative or very low ANX7 lates with high levels of ANX7 protein (Fig. 3). Thus, the weak expression), survival is up to 95% (P ϭ 0.014, log-rank test; immunohistochemical reaction for ANX7 in nonmetastatic cells Breslow-Gehan-Wilcoxon test; Tarone-Ware; Peto-Peto-Wilc- and tumors appears to represent a truly low level of ANX7 oxon and Harrington-Fleming). protein that has in vitro parallels. These results therefore indi- Cytoplasmic ANX7 Expression Is Associated with cate that high ANX7 expression is associated with the most BRE-2 Grade and HER2-Negative Patients. Parallel sec- aggressive types of breast cancer. tions of the same specimens were investigated for alteration in the Prognostic Impact of ANX7 Expression. To evaluate expression of ER, PR, p53, and HER2 proteins (13). ANX7 was the prognostic significance of ANX7, we have used a tissue negative or weakly positive in normal glands adjacent to the cancer microarray (12) containing 552 breast tumor tissue specimens. on individual locations in this tissue microarray and benign glands Each sample is accompanied by clinical follow-up data for up to that were occasionally present adjacent to the cancer tissue. In a 105 months. The samples on the array were evaluated for ANX7 different study on a separate human tumor tissue microrray, we expression by immunohistochemistry. The presence of ANX7 in were able to analyze ANX7 expression in four normal glands. We

Table 2 Multivariate analyses A. Cox regression using all cases on the array Range or 95% Confidence interval Parameter category Added risk for added risk Significance BRE grade 1–3 1.901 (1.356–2.666) 0.0002 pN 1&2vs. 0 4.096 (2.455–6.834) Ͻ0.0001 pT 4 vs. 1, 2, & 3 2.562 (1.577–4.160) 0.0001 HER2 0–3 1.316 (1.089–1.590) 0.004 PR Pos. vs. Neg. 1.678 (0.994–2.833) 0.053 ER Pos. vs. Neg. 1.205 (0.684–2.121) 0.519 p53 Pos. vs. Neg. 1.437 (0.934–2.212) 0.099 ANX7 0–3 1.306 (0.969–1.762) 0.080 B. Cox regression using only HER2-negative (HER2 ϭ 0 or 1) cases (87.1% of the cases) Range or 95% Confidence interval Parameter category Added risk for added risk Significance BRE grade 1–3 1.955 (1.3333–2.869) 0.001 pN 1&2vs. 0 3.988 (2.253–7.060) Ͻ0.0001 pT 4 vs. 1, 2, & 3 3.230 (1.875–5.567) Ͻ0.0001 HER2 0–3 2.061 (1.174–3.620) 0.012 PR Pos. vs. Neg. 2.274 (1.272–4.065) 0.006 ER Pos. vs. Neg. 1.202 (0.619–2.332) 0.587 p53 Pos. vs. Neg. 1.238 (0.758–2.023) 0.394 ANX7 0–3 1.590 (1.109–2.281) 0.012 C. Cox regression using only HER2 ϭ 0 cases (74.8% of the cases) Range or 95% Confidence Parameter category Added risk interval for added risk Significance BRE grade 1–3 2.236 (1.400–3.571) 0.001 pN 1&2vs. 0 4.876 (2.400–9.905) Ͻ0.0001 pT 4 vs. 1, 2, & 3 2.849 (1.488–5.456) 0.002 PR Pos. vs. Neg. 1.578 (0.793–3.120) 0.195 ER Pos. vs. Neg. 1.253 (0.565–2.780) 0.579 p53 Pos. vs. Neg. 1.379 (0.778–2.431) 0.278 ANX7 0–3 1.996 (1.306–3.049) 0.001

found negative or, at best, weakly positive cyto-immunolabeling lacking detectable HER2 expression. Cox regression analysis for ANX7 (data not shown). Table 1 describes the composite reveals that even after adjusting for ER, PR, p53, pT, pN, and univariate analysis of all 553 patients in terms of classical clinico- BRE grade 2, HER2-negative patients suffer a doubling in the pathological risk factors, including nodal status, tumor grade, and risk of death with each increasing level of ANX7 expression. stage. We include in Table 1 the known prognostic factors such as Remarkably, in HER2-negative patients, the difference in risk is p53, HER2, ER, and PR. Also shown in this table are the unad- 10-fold between those with negative ANX7 expression and justed Ps for the log-rank test of homogeneity of strata (shown those with strong ANX7 expression. The clinical treatment of separately for each variable evaluated), as well as the strata com- primary breast cancers has been greatly complicated by the pared by each test. Based on the analysis of all of the parameters, inability to accurately predict which tumors will eventually it is evident that high cytoplasmic ANX7 expression has a signif- become invasive and metastatic, and which will become local- icant and specific impact on the probability of survival for patients ized and indolent. Strong expression of HER2 in 20–35% of the with BRE-2 grade tumors (Fig. 5A; P ϭ 0.001) or for patients breast cancer patients is known to be associated with poor whose tumors do not express HER2 (Fig. 5B, P ϭ 0.002). For prognosis and has been used to predict response to treatment example, in the BRE-2 patient cohort, 100% of the patients with with the anti-HER2 antibody trastuzumab (Herceptin). Our data very low ANX7 expression survived. By contrast, only 52% of therefore suggest that the expression level of ANX7 can help to those with strong ANX7 expression survived. stratify the remaining HER2-negative patients who need the Multivariate Analysis Showing ANX7 as a Risk Bi- most focused attention. At a minimum, the value of our result omarker for HER2-Negative Patients. We have performed for breast carcinomas without HER2 expression is that the multivariate analyses (Cox regression, Table 2) on the data to ANX7 gene assay might provide a simple and reliable survival determine the significance and independence of the ANX7 parameter for clinicians to include in patient management plans immunoassay data in predicting the outcome and progression of for early detection and treatment options. breast cancer. All of the tissues were from resected breast To our knowledge, this is the first report on ANX7 protein cancers without any preceding therapy that could have con- expression in a large series of patients with breast cancer. founded the results. At the time of sample collection, neoadju- vant therapy of breast cancer was not performed at University Hospital of Basel. We used traditional variables in the multivariate analyses including the tumor stage (pT), nodal status, pN, and BRE grade as assessed from the medical records of the donor patients. In addition, we also added immunohistochemical evaluations for HER2, PR, ER, p53, and ANX7, which we carried out on the identical tissue microarray samples. As shown in Table 2A, these analyses show that for the entire cohort, the level of ANX7 has a marginally significant value (P ϭ 0.08; added risk ϭ 1.3; 95% confidence interval, 0.9–1.8) as a prognostic indicator. However, when we looked only at the subpopu- lation of patients with low HER2 levels (HER2 ϭ 0 or 1, Table 2B), which comprises 87.1% of the cohort, we found that the level of ANX7 expression has a definitely significant prognostic value even after considering the effects of all of the other variables in the equation. Specifically, the ANX7 level is associated with an increased risk of 1.6 (95% confidence interval, 1.1–2.3) and has a significance of P ϭ 0.012 as a prognostic marker. This finding is even more pronounced with a significance of P ϭ 0.001 when we look only at the HER2 ϭ 0 patients (about 75% of the cohort; Table 2C), for whom the risk is doubled for each successive step of ANX7 level (Fig. 6). This increased risk with ANX7 in the HER2-negative cohort is comparable with the increased risk associated with BRE grade in the entire cohort population. These results indicate that ANX7 levels have considerable potential for early detection of breast tumors, giving patients and physicians a new tool for managing breast cancer.

DISCUSSION Fig. 6 Relative risk of death as a function of ANX7 expression. The The results obtained from 1077 breast tissue specimens relative risk of death is doubled for each successive step of ANX7 level, where 0 represents very weak ANX7 expression, 1 and 2 represent a show that increased ANX7 expression is associated with meta- moderate level of ANX7 expression, and 3 represents a high level of static disease and significantly decreased survival in those breast ANX7 expression. This value was adjusted for pT, nodal status, pN, cancer patients who present with BRE-2 grade tumors or tumors BRE grade, HER2, progesterone receptor, estrogen receptor, and p53.

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Meera Srivastava, Lukas Bubendorf, Mark Raffeld, et al.

Clin Cancer Res 2004;10:2344-2350.

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