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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 5  |  Page : 68-73

Serum biomarker 3144 m/z for prognostic detection in Chinese postmenopausal breast cancer patients


1 Department of Cancer Research Institute, Zhejiang Key Laboratory of Diagnosis and Treatment Technology on Thoracic Oncology (Lung and Esophagus), Zhejiang Cancer Hospital, Zhejiang Cancer Research Institute, Hangzhou, Zhejiang, China
2 Department of Medical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China
3 Department of Breast Cancer Surgery, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, China

Date of Web Publication31-Aug-2015

Correspondence Address:
Dr. Xiaojia Wang
Zhejiang Cancer Hospital, No. 38, Guangji Road, Banshan Bridge, Hangzhou, Zhejiang
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.163844

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 > Abstract 

Context: Breast cancer becomes more prevalent with advancing age. The increased risk of breast cancer needs to be considered when choosing a treatment plan and a kind of detection method for the postmenopausal woman. Better breast cancer prognostication may improve selection of patients for adjuvant therapy.
Aims: The aim of this study is to investigate the role of serum protein peak 3144 m/z in postmenopausal breast cancer patients, whether if it could be used as a potential prognostic tool.
Settings and Design: Two hundred and two postmenopausal breast cancer patients were involved in this retrospective study at Zhejiang Cancer Hospital.
Subjects and Methods: Serum level of protein peak 3144 m/z was assessed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.
Statistical Analysis Used: Prognostic factors were compared across subgroups of patients depending on the protein peak 3144 m/z levels by Chi-square test. The log-rank test was used to compare survival curves, and Cox proportional hazards regression analysis was performed to identify prognostic factors.
Results: The percentage of cases with higher 3144 m/z protein peak was 32.7% (66/202) in postmenopausal breast cancer patients. The serum protein peak 3144 m/z was positively related to lymph node metastasis. Patients with higher protein peak 3144 m/z had significantly poorer overall survival compared with patients with lower serum protein peak 3144 m/z (P = 0.0053). Multivariate regression analysis also revealed that protein peak 3144 m/z was an independent prognostic factor in postmenopausal breast cancer patients (borderline, P = 0.064).
Conclusions: The protein peak 3144 m/z was a potential prognostic factor, and it could be used as a prognostic monitoring tool in postmenopausal breast cancer patients.

Keywords: Blood proteins, breast neoplasms, postmenopause, prognosis


How to cite this article:
Gao Y, Xu S, Cao W, Chen Z, Wang X, Zou D. Serum biomarker 3144 m/z for prognostic detection in Chinese postmenopausal breast cancer patients. J Can Res Ther 2015;11, Suppl S1:68-73

How to cite this URL:
Gao Y, Xu S, Cao W, Chen Z, Wang X, Zou D. Serum biomarker 3144 m/z for prognostic detection in Chinese postmenopausal breast cancer patients. J Can Res Ther [serial online] 2015 [cited 2019 Sep 21];11:68-73. Available from: http://www.cancerjournal.net/text.asp?2015/11/5/68/163844


 > Introduction Top


Breast cancer is an invasive and ultimately fatal disease that gradually increased in China over the last few decades, and it is at present the most commonly diagnosed neoplasm among women. In addition, despite the substantial progress made in cancer therapy, breast cancer is the second leading cause of female cancer deaths, following lung cancer. [1],[2] The main prognostic factors currently used to determine eligibility for administration of adjuvant systemic therapy include both clinical and pathological parameters, e.g., patient's age at diagnosis, hormone receptor status, lymph node status, grade of malignancy. [3],[4] Young age (<35) was proved to be an independent predictor for the poor outcome of breast cancer, [5] and hormone receptor status is also a predictor of prognosis. [6] However, there are 30-50% of breast cancer patients who received appropriate loco-regional treatment and adjuvant systemic therapy will develop metastatic relapse and die, while there are a substantial percentage of patients that would have survived without chemotherapy and hormonal therapy. [7] Currently, applied prognostic markers do not suffice for precise risk-group determination in breast cancer. As we known, young premenopausal women with breast carcinomas have an overall worse prognosis than older, postmenopausal women. [8],[9] There were lots of studies focus on searching prognostic factors for premenopausal patients, [10] but the research concerning on those postmenopausal breast cancer patients is few. The postmenopausal breast cancer patients need to be paying more attentions.

In the search for these markers, evaluation of protein expression signature in body fluids can be used to discover new noninvasive biomarkers for measurement and detections of breast cancer. Investigators have identified serum biomarkers include cancer antigen 153 (CA153) and carcinoembryonic antigen previously. [11] Although these proteins are useful for monitoring ongoing treatment in breast cancer patient, the great heterogeneity of breast cancer suggests that more than one biomarker is need for different subtype breast cancer patients. A promising approach to biomarker discovery in proteomic technologies is the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). By combining chromatography with laser desorption/ionization MS, this platform has enabled high-throughput mass profiling of highly complex biological samples, such as tissue lysates and serum. In previous study, we found that the protein peak 3144 m/z was higher expressed in high-metastasis ovarian cancer cell line than it in low-metastasis cell line's. [12] That result was validated in the serum of advanced ovarian cancer and gastric cancer patients. [12],[13] However, the possible role of the protein peak 3144 m/z in postmenopausal breast cancer is not very clear.

In present study, we aimed to evaluate serum protein peak 3144 m/z could be used as a potential prognostic tool for postmenopausal breast cancer, taking into account clinic-pathological features.


 > Subjects and Methods Top


Patients

Two hundred and two postmenopausal breast cancer patients from June 2006 to December 2009 were involved in this retrospective study at Zhejiang Cancer Hospital. Menopausal status of the cases was obtained through examination of the medical records. The histopathological type, lymph node metastasis, and hormone receptor status of the cases were determined by pathologists. All patients underwent conventional postsurgical treatment (chemotherapy, radiotherapy, endocrinotherapy and target therapy) and the serum samples were collected before treatment. All serum samples were obtained with medical ethics approval from Independent Medicine Ethics Committee at Zhejiang Cancer Hospital. The study was undertaken in accordance with the ethical standards of the World Medical Association Declaration of Helsinki. Age, body weight index, menarche age, pregnancy times, abortion history, family history, and blood type were obtained from the medical records. Lymph node involvement metastasis, estrogen receptor and progesterone receptor status and HER-2/neu expression were diagnosed by pathological examination. The follow-up was performed by telephone or mail after surgery. The endpoint of the study was overall survival (OS). OS was calculated as the period from the date of diagnosis to the date of death or the date of last follow-up.

Sample preparation

Peripheral blood samples were collected from fasting breast cancer patients before surgery, and stored at 4°C. Within 2 h, the blood samples were centrifuged at 4000 r/min for 5 min at 4°C to isolate the serum. The serum samples were then centrifuged at 14,000 r/min for 5 min at 4°C to remove all residual cell debris. The samples were transferred into a new centrifuge tube on ice and stored at −80°C. Before use, the serum samples were placed on the ice water bath to increase the temperature to 0°C. In a tagged 1.5 ml centrifuge tube containing 20 μl 9 mol/L buffer (urea, 9 mol/L; 2% CHAPS; Tris-HCl, 50 mmol/L; 1% DTT; pH = 9.0), 10 μl serum was added. The diluted sample was placed at room temperature for 10 min. Then 360 μl binding buffer was added and mixed well.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry analysis

Weak cation-exchange nanobeads were used to enrich serum protein. All elution steps were performed in accordance with annexed protocols by the manufacturer. Each sample (1 μl) was added to an Au chip and then was tested after the chip was dry. Phosphate buffered saline-C mass spectrometer was used to read the chip information. Before data collection, the instrument was calibrated with the all-in-one standard protein NP20 chip calibration to within 0.1%. The instrument settings were as follows: Laser intensity, 175; detection sensitivity, 8; and optimized molecular weight range, 1000-15000 Da. For each point, data were collected 90 times. Ciphergen ProteinChip 3.2.1 software (Ciphergen Biosystems, Freemont, CA) was used for data collection. On the basis of the receiver operating characteristic curve for the differentially expressed protein (3144 m/z) that we obtained from patients with advanced ovarian cancer, when the boundary value is 1.15, the sensitivity and specificity would be 65.4% and 91.4%, respectively. Therefore, the boundary value 1.15 was used to evaluate the level of serum protein peak 3144 m/z.

Statistical analysis

Chi-square test was used to test whether any relationship between the intensity of the peak and the clinicopathologic features. Survival curves were plotted using the Kaplan-Meier method, and survival differences between groups were determine using the log-rank test. Hazard ratios (HR) and associated 95% confidence intervals (95% CIs) were estimated using a Cox regression proportional hazard model. P < 0.05 was considered statistically significant.


 > Results Top


Correlation between serum protein peak 3144 m/z and patient clinical pathologic factors

The percentage of cases with higher 3144 m/z protein peak was 32.7% (66/202) in postmenopausal breast cancer patients when the boundary value 1.15 was used as cutoff to evaluate the expression level of serum protein peak 3144 m/z.

Patients with higher serum protein peak 3144 m/z levels tend to have a higher proportion of tumor with lymph node metastasis [Table 1]. The percentage of patients owned higher 3144 m/z protein peak level with lymph node metastasis was significantly higher than those of patients without lymphatic metastasis (68.2% vs. 48.5%, P = 0.006). There were no significant correlations between serum protein peak 3144 m/z and other clinicopathological features, such as hormone receptor status, clinical stage, menarche age, and parity.
Table 1: Association analyses between the level of serum protein peak 3144 m/z and clinicopathologic factors in 202 postmenopausal breast cancer patients


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Prognostic significance of serum protein peak 3144 m/z

As determined using Kaplan-Meier method and the log-rank test, the high level of serum protein peak 3144 m/z was correlated with a shorter OS of postmenopausal breast cancer patients (P = 0.0053). Postmenopausal breast cancer patient with a higher level of serum protein peak 3144 m/z was associated with poor prognosis [Figure 1]. The median OS of patients with higher serum protein peak 3144 m/z was 48 months while patients with lower serum protein peak 3144 m/z was 53 months [Table 2].
Figure 1: Kaplan-Meier curves for overall survival of prognosis in 202 postmenopausal breast cancer patients according to the categories of low and high level of serum protein peak 3144 m/z

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Table 2: Univariate survival analysis of OS in 202 postmenopausal breast cancer patients


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In univariate analysis, serum protein peak 3144 m/z (HR = 0.333, 95% CI = 0.148 ~ 0.751), lymph node metastasis (HR = 0.099, 95% CI = 0.023 ~ 0.420) and HER-2 status (HR = 0.253, 95% CI = 0.075 ~ 0.848) were significant prognostic factors for OS (P < 0.05). Serum protein peak 3144 m/z was a significant prognostic factor for OS (P = 0.0008). Multivariate analysis indicated that serum protein peak 3144 m/z was a board line prognostic factor for OS (P = 0.064). Positive lymph node involved was the only independent prognostic factor from the result of Cox regress analysis [Table 3].
Table 3: Univariate and multivariate Cox regression analysis for various potential prognostic factors of OS in 202 postmenopausal breast cancer patients


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 > Discussion Top


Breast cancer becomes more prevalent with advancing age. The increased risk of breast cancer in postmenopausal woman needs to be considered when choosing a treatment plan or a kind of prognostic surveillance. Traditionally, the prognosis of breast cancer has been known to be associated with clinical stage and molecular subtype, and the number of positive lymph nodes is a key indicator of prognosis in breast cancer patients. [14],[15] Result of our study showed the serum protein peak 3144 m/z was closely related to positive lymph node metastasis, representing relatively high percentages of higher expression the serum protein peak 3144 m/z was in lymph node positive patients, when compared with lymph node negative. Lymph node metastasis has been used as an independent prognostic factor for clinical treatment and surveillance. Nevertheless, judgment on involved lymph nodes depends on the surgical and pathologic procedure, considered the accuracy and efficiency of methods to identify lymph node metastasis, serum biomarker is a safer and more convenience supplementary measure. The interrelation between serum protein peak 3144 m/z and positive lymph node indicated serum protein peak 3144 m/z was a proper substitute superior to positive lymph nodes as a prognostic surveillance biomarker.

Serum is the most readily available sample type for clinical testing because it can be easily collected and is available at all stages of the disease. SELDI-TOF is a novel technique used in proteomics research directed at the identification of new tumor protein markers in the serum. [16] We have previously used SELDI-TOF to compare the difference between high- and low- metastasis ovarian cancer cell lines, screening a new metastasis-related protein peak 3144 m/z and validating protein peak 3144 m/z expression in patients with advanced ovarian cancer and gastric cancer. [12],[13] In the current study, we investigated sera of 202 postmenopausal breast cancer patients obtained after diagnosis, but prior to the administration of surgery, adjuvant therapy, in search for the specific prognostic biomarker. The current study showed that the percentage of cases with higher 3144 m/z protein peak was 32.7% (66/202) in postmenopausal breast cancer patients. The sensitivity of CA153, a commonly used serum biomarker for monitoring metastasis and recurrence in breast cancer, was only 15.3% to 22.5%. [17],[18] The sensitivity of serum protein peak 3144 m/z is higher than that of traditional CA153 in breast cancer patients. The serum protein peak 3144 m/z will be a valuable candidate prognostic marker.

Although the results of Chi-square test showed there were no significant correlations between serum protein peak 3144 m/z and clinicopathological features, such as hormone receptor status, molecular subtype, clinical stage, pregnancy times, and abortion history, but the survival analysis and univariate analysis results indicated that high level of serum protein peak 3144 m/z and positive lymph node involved both were predictor for poor outcome in breast cancer. The present study underscores the prognostic role of serum protein peak 3144 m/z, and the association to clinical-pathologic features in postmenopausal breast cancer patients. As shown from the Kaplan-Meier survival curves in [Figure 1], it can be seen that patients' survival rates were significantly decreased in patients with high level of serum protein peak 3144 m/z. The high level of serum protein peak 3144 m/z was correlated with a shorter OS of postmenopausal breast cancer patients. Cox regression analysis revealed that lymph node involvement was an independent significant prognostic factor, which was consistent with many reports. [19],[20],[21],[22] Lymph nodes metastasis is the most important prognostic factor in patients with operable breast cancer. [20],[21] The current study results also proof this opinion. Based on our results, the prognostic value of serum protein peak 3144 m/z is worth to further research in largely postmenopausal breast cancer patients. However, the significance of protein peak 3144 m/z remains weak due to the lack of validation, just as many other serum biomarkers for breast cancer by SELDI-TOF MS or MALDI-TOF MS. [23],[24]

To prepare for the validation, we have queried the SwissProt database (http://expasy.org/tools/tagident.html) using molecular weight and found the 3144 m/z protein peak to be CD24, mucin-like adhesion molecule belonging to a group of glycosyl-phosphatidylinositol anchor proteins. CD24 was found to be highly expressed in breast cancer, nonsmall cell lung cancer, gastrointestinal cancer, and other tumor tissue. [25],[26],[27] CD24 may be involved in the development of tumors by promoting tumor cell proliferation, invasion, and metastasis. [27] It has been reported that CD24 expression was a poor prognosis marker in hormone receptor positive breast cancer. [28],[29] Though CD24 appears to be a protein marker for tumor invasion, metastasis, and prognosis, the relationship between CD24 and 3144 m/z protein peak need further study to confirm. However, this finding has to be verified because different proteins may have similar protein weights. Further validation of the protein peak 3144 m/z can help to facilitate the prognosis of breast cancer, as well as enable development of absolute quantitative assays. Although it is yet to be verified, the 3144 m/z protein peak provides a novel biomarker for the screening of prognosis in postmenopausal breast cancer patients.


 > Conclusion Top


The present study showed that the protein peak 3144 m/z was a poor prognostic factor in postmenopausal breast cancer patients. Further prospective and longitudinal studies are needed to evaluate whether serum protein peak 3144 m/z could be used as a prognostic tool in postmenopausal breast cancer patients monitoring and management.

Acknowledgments

This work was supported by Health and Family Planning Commission of Zhejiang Province (2011KYA033), and Science Technology Department of Zhejiang Province (2013C33205).

Financial support and sponsorship

Health and Family Planning Commission of Zhejiang Province [2011KYA033], and Science Technology Department of Zhejiang Province [2011RCA014].

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Chen WQ, Zheng RS, Zeng HM, Zhang SW, Li GL, Wu LY, et al. Incidence and mortality of breast cancer in China, 2008. Thorac Cancer 2013;4:59-65.  Back to cited text no. 1
    
2.
Zeng H, Zheng R, Zhang S, Zou X, Chen W. Incidence and mortality of female breast cancer in China, 2009. Thorac Cancer 2013;4:400-4.  Back to cited text no. 2
    
3.
Rakha EA, Ellis IO. Modern classification of breast cancer: Should we stick with morphology or convert to molecular profile characteristics. Adv Anat Pathol 2011;18:255-67.  Back to cited text no. 3
    
4.
Patani N, Martin LA, Dowsett M. Biomarkers for the clinical management of breast cancer: International perspective. Int J Cancer 2013;133:1-13.  Back to cited text no. 4
    
5.
Yildirim E, Dalgiç T, Berberoglu U. Prognostic significance of young age in breast cancer. J Surg Oncol 2000;74:267-72.  Back to cited text no. 5
    
6.
Higa GM, Fell RG. Sex hormone receptor repertoire in breast cancer. Int J Breast Cancer 2013;2013:284036.  Back to cited text no. 6
    
7.
Polychemotherapy for early breast cancer: An overview of the randomised trials. Early Breast Cancer Trialists′ Collaborative Group. Lancet 1998;352:930-42.  Back to cited text no. 7
[PUBMED]    
8.
Tichy JR, Lim E, Anders CK. Breast cancer in adolescents and young adults: A review with a focus on biology. J Natl Compr Canc Netw 2013;11:1060-9.  Back to cited text no. 8
    
9.
Tricoli JV, Seibel NL, Blair DG, Albritton K, Hayes-Lattin B. Unique characteristics of adolescent and young adult acute lymphoblastic leukemia, breast cancer, and colon cancer. J Natl Cancer Inst 2011;103:628-35.  Back to cited text no. 9
    
10.
Talley L, Chhieng DC, Bell WC, Grizzle WE, Frost AR. Immunohistochemical detection of EGFR, p185(erbB-2), Bcl-2 and p53 in breast carcinomas in pre-menopausal and post-menopausal women. Biotech Histochem 2008;83:5-14.  Back to cited text no. 10
    
11.
Chen Y, Zheng YH, Lin YY, Hu MH, Chen YS. Clinical and prognostic significance of preoperative serum CA153, CEA and TPS levels in patients with primary breast cancer. Zhonghua Zhong Liu Za Zhi 2011;33:842-6.  Back to cited text no. 11
    
12.
Zheng ZG, Gao Y, Linhui G, Hanzhou M. Discovery of metastasis-associated biomarkers in ovarian cancer using SELDI-TOF: An in vitro and clinical study. Clin Oncol Cancer Res 2009;6:317-21.  Back to cited text no. 12
    
13.
Xu SH, Su D, Zhu X, Zheng ZG, Xu Q, Zhu CH, et al. Expression of a protein peak (3144 m/z) in serum and its clinical significance in patients with gastric cancer. Zhonghua Wei Chang Wai Ke Za Zhi 2012;15:165-8.  Back to cited text no. 13
    
14.
Vinh-Hung V, Verkooijen HM, Fioretta G, Neyroud-Caspar I, Rapiti E, Vlastos G, et al. Lymph node ratio as an alternative to pN staging in node-positive breast cancer. J Clin Oncol 2009;27:1062-8.  Back to cited text no. 14
    
15.
Wu SG, He ZY, Li Q, Sun JY, Li FY, Lin Q, et al. Prognostic value of metastatic axillary lymph node ratio for Chinese breast cancer patients. PLoS One 2013;8:e61410.  Back to cited text no. 15
    
16.
Jiang M, Gu G, Ni B, Wang W, Shi J, Liao P, et al. Detection of serum protein biomarkers by surface-enhanced laser desorption/ionization in patients with adenocarcinoma of the lung. Asia Pac J Clin Oncol 2013;10:e7-12.  Back to cited text no. 16
    
17.
Molina R, Auge JM, Farrus B, Zanon G, Pahisa J, Munoz M, et al. Prospective evaluation of carcinoembryonic antigen (CEA) and carbohydrate antigen 15.3 (CA 15.3) in patients with primary locoregional breast cancer. Clin Chem 2010;56:1148-57.  Back to cited text no. 17
    
18.
Gion M, Mione R, Leon AE, Dittadi R. Comparison of the diagnostic accuracy of CA27.29 and CA15.3 in primary breast cancer. Clin Chem 1999;45:630-7.  Back to cited text no. 18
    
19.
Demircioglu F, Demirci U, Kilic D, Ozkan S, Karahacioglu E. Clinical significance of lymph node ratio in locally advanced breast cancer molecular subtypes. Onkologie 2013;36:637-40.  Back to cited text no. 19
    
20.
Osako T, Tsuda H, Horii R, Iwase T, Yamauchi H, Yagata H, et al. Molecular detection of lymph node metastasis in breast cancer patients treated with preoperative systemic chemotherapy: A prospective multicentre trial using the one-step nucleic acid amplification assay. Br J Cancer 2013;109:1693-8.  Back to cited text no. 20
    
21.
Sestak I, Dowsett M, Zabaglo L, Lopez-Knowles E, Ferree S, Cowens JW, et al. Factors predicting late recurrence for estrogen receptor-positive breast cancer. J Natl Cancer Inst 2013;105:1504-11.  Back to cited text no. 21
    
22.
Donepudi MS, Kondapalli K, Amos SJ, Venkanteshan P. Breast cancer statistics and markers. J Cancer Res Ther 2014;10:506-11.  Back to cited text no. 22
    
23.
Böhm D, Keller K, Wehrwein N, Lebrecht A, Schmidt M, Kölbl H, et al. Serum proteome profiling of primary breast cancer indicates a specific biomarker profile. Oncol Rep 2011;26:1051-6.  Back to cited text no. 23
    
24.
Opstal-van Winden AW, Krop EJ, Kåredal MH, Gast MC, Lindh CH, Jeppsson MC, et al. Searching for early breast cancer biomarkers by serum protein profiling of pre-diagnostic serum; a nested case-control study. BMC Cancer 2011 26;11:381.  Back to cited text no. 24
    
25.
Buck K, Hug S, Seibold P, Ferschke I, Altevogt P, Sohn C, et al. CD24 polymorphisms in breast cancer: Impact on prognosis and risk. Breast Cancer Res Treat 2013;137:927-37.  Back to cited text no. 25
    
26.
Karimi-Busheri F, Rasouli-Nia A, Zadorozhny V, Fakhrai H. CD24+/CD38 - As new prognostic marker for non-small cell lung cancer. Multidiscip Respir Med 2013;8:65.  Back to cited text no. 26
    
27.
Jiao XL, Zhao C, Niu M, Chen D. Downregulation of CD24 inhibits invasive growth, facilitates apoptosis and enhances chemosensitivity in gastric cancer AGS cells. Eur Rev Med Pharmacol Sci 2013;17:1709-15.  Back to cited text no. 27
    
28.
Kim HJ, Kim MJ, Ahn SH, Son BH, Kim SB, Ahn JH, et al. Different prognostic significance of CD24 and CD44 expression in breast cancer according to hormone receptor status. Breast 2011;20:78-85.  Back to cited text no. 28
    
29.
Horiguchi K, Toi M, Horiguchi S, Sugimoto M, Naito Y, Hayashi Y, et al. Predictive value of CD24 and CD44 for neoadjuvant chemotherapy response and prognosis in primary breast cancer patients. J Med Dent Sci 2010;57:165-75.  Back to cited text no. 29
    


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