Tumor Biol. DOI 10.1007/s13277-014-1875-3

RESEARCH ARTICLE

Clinical significance of serum tenascin-C levels in breast cancer D. Tastekin & F. Tas & S. Karabulut & D. Duranyildiz & M. Serilmez & M. Guveli & S. Vatansever

Received: 19 January 2014 / Accepted: 19 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Tenascin-C (TNC) is a key molecule in tissue remodeling, and high levels are observed in many diseases, including heart failure, thrombosis, atherosclerosis, and cancer. High TNC expression by immunohistochemical analysis has been shown in invasive and metastasizing tissues from a variety of cancers, including colon, lung, brain, and breast. This study was conducted to investigate the serum level of TNC in breast cancer patients and its relationship with tumor progression and known prognostic parameters. Ninety-six breast cancer patients were enrolled into the study. Serum samples were obtained on first admission before adjuvant and metastatic treatments were given and at follow-up. Serum TNC levels were determined by the solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) method. Median age of diagnosis was 48 years old (range, 29–80). Thirtyseven (39 %) patients had metastatic breast cancer. The mean TNC levels were found to be significantly higher in patients with breast cancer (344.1±42.4 pg/mL) compared to those in healthy controls (137.2±26.8 pg/mL) (p=0.005). Serum TNC level in grade 3 tumors was found to be significantly higher than in grades 1–2 tumors (p=0.04). No correlation was detected between serum TNC levels and other prognostic parameters analyzed, including presence of metastasis, lymph node involvement, and tumor size. Serum TNC level had no significantly adverse effect on survival in univariate and D. Tastekin (*) : F. Tas : S. Karabulut : S. Vatansever Department of Medical Oncology, Oncology Institute, Istanbul University, Capa, 34390 Istanbul, Turkey e-mail: [email protected] D. Duranyildiz : M. Serilmez Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey M. Guveli Department of Radiation Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey

multivariate analyses (p=0.65 and p=0.85, respectively). In conclusion, although serum TNC levels are elevated, it has no predictive or prognostic roles on survival in breast cancer patients. Keywords Tenascin-C . Breast cancer . Prognosis

Introduction Tenascin-C (TNC), a rather “old” molecule, discovered more than 30 years ago, is a key factor in tissue remodeling, and its deregulated high expression is causally linked to many diseases, including heart failure, thrombosis, atherosclerosis, and cancer. It is considered to have an impact on the microenvironment through direct interactions with cells and growth factors, thus regulating cell responses such as cell survival, migration, proliferation, and transdifferentiation [1]. High TNC expression has been shown in invasive and metastasizing tissues from a variety of cancers, including colon, lung, brain, and breast [2–4]. In addition, TNC expression is linked to lymph node metastasis in breast, colon, liver, and oral squamous cell carcinoma [5]. Many reports suggest a supportive role for TNC in tumor growth, metastasis, tumor angiogenesis, and inhibition of immune surveillance [6]. In a mouse model, TNC knockdown causes dramatic inhibition of lung metastasis and colonization by breast cancer cells [7]. TNC has been subjected to extensive investigation in the breast due to its selective expression, where it is tightly regulated in the normal resting breast but highly induced in breast cancer. In the breast, TNC expression in the invasion border was a significant prognostic factor for local recurrence and was independently linked with lymph node metastasis, suggesting that TNC could play a role in metastasis formation [8]. Although TNC was proposed as a stromal marker in breast cancer, it was demonstrated to be expressed by both

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transformed epithelial cells and stromal [5]. We aimed in this study to investigate serum TNC levels with regard to prognostic factors in patients with breast cancer.

Materials and methods Patients The study group consisted of patients consecutively presenting to the Institute of Oncology, University of Istanbul. Ninety-six pathologically verified breast cancer patients were investigated. Diagnosis of breast cancer was histologically proved either with tru-cut biopsy of the primary lesion or metastatic site and staged according to the sixth edition of AJCC. For histological evaluation, tissue sections (2 mm) were deparaffinized and stained using hematoxylin and eosine. Grading of tumors was established according to the modified Bloom-Richardson grading system. Estrogen receptor (ER), progesterone receptor (PR), and HER2 status were evaluated in the sample sections using appropriate antibodies. The immunohistochemical staining was assessed upon visual inspection with an optical microscope and considered as positive if the percentage of cells stained positive was more than 5 %. In case of 2(+) staining by IHC, HER2 gene amplification was analyzed by fluorescence in situ hybridization (FISH). For comparison of serum levels of TNC, age- and sexmatched 28 healthy women controls were included in the analysis. Institutional review board approval was obtained from each subject prior to the commencement of the study. Measurement of serum TNC levels Serum samples were obtained on first admission before any adjuvant and metastatic treatments were given or follow-up patients. Blood samples were obtained from patients with breast cancer and healthy controls by venipuncture and clotted at room temperature. The sera were collected following centrifugation and frozen immediately at −20 °C until analysis. Serum TNC levels were determined by the solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) method. The TNC ELISA (USCN, China) uses a double-antibody sandwich ELISA to determine the level of TNC in the samples. Serum samples and standards are added to the wells which are precoated with human TNC monoclonal antibody. Following incubation, TNC antibodies labeled with biotin and combined with streptavidin-HRP are added to form an immune complex and allowed to incubate for 1 h. Unbound material is washed away, and then a chromogen solution is added for the conversion of the colorless solution to a blue solution, the intensity of which is proportional to the amount of TNC in the sample. As the effect of the acidic stop solution,

Table 1 Characteristics of the patient and disease Variables

n (%)

No. of patients Age, years Median (range) 48 (29–80) −49/50+ Grade 1/2/3/unknown Ki-67a 0–14/15–30/>30 ER −/+/unknown PR −/+/unknown HER2 −/+/unknown Classificationa Luminal/HER2+/Triple (−)/Triple (+) T statusb 1/2/3/4/unknown N statusb −/+/unknown M status 0/1 Serum LDH level (450 U/L)a Normal/elevated Serum CA15-3 level (35 U/mL)a Normal/elevated Response to chemotherapyc Yes/no/unknown

96 (100)

Last status Alive/dead

51 (53)/45 (47) 4 (4)/26 (27)/24 (25)/42 (44) 11 (18)/14 (24)/34 (58) 27 (28)/68 (71)/1 (1) 32 (33)/63 (66)/1 (1) 64 (67)/31 (32)/1 (1) 56 (58)/18 (19)/8 (8)/13 (14)

18 (30)/21 (36)/3 (5)/3 (5)/14 (24) 23 (39)/23 (39)/13 (22) 59 (61)/37 (39) 69 (73)/26 (27) 68 (72)/26 (28) 46 (78)/10 (17)/3 (5) 80 (83)/16 (17)

a

Patients with unknown data concerning the variables are not included in the analysis b In 59 nonmetastatic diseases c In 37 metastatic and 22 locally advanced diseases

the color has become yellow. The colored reaction product is measured using an automated ELISA reader (Rayto, RT1904C Chemistry Analyzer, Atlanta, GA, USA). The results were expressed as picograms per milliliter.

Table 2 The values of tenascin-C in patients with breast cancer and healthy controls Assay

Patients (n=96) Controls (n=30) p value Mean (±SE) Mean (±SE)

Tenascin-C level (pg/mL) 344.1 (42.4)

137.2 (26.8)

Significant p value (less than 0.05) is highlighted in italics

0.005

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Statistical analysis Continuous variables were categorized using median values as cutoff point. Assessment of relationships and comparisons between various clinical/laboratory parameters and serum levels of TNC assays were accomplished using the MannWhitney U test. Spearman’s rank order correlation was used for correlation analysis. Survival was calculated from the date of first admission to hospital to death resulting from any cause or to last contact with the patient or any family member. The Kaplan-Meier method was used for the estimation of survival of patient, and differences in survivals were assessed by the log-rank statistic. A p value of mean) 32.3 (1.9) Low (30 ER Negative Positive PR Negative Positive HER2 Negative Positive Classification Luminal Others Classification Triple positive Others Classification Triple negative Others Classification HER2 enriched Others T status T1 T2–T4 N status Negative Positive M status Negative Positive Serum LDH level

18 (13) 78 (9) 78 (6)

0.65

Significant p values (less than 0.05) are highlighted in italics

was associated with a higher proliferation rate measured by Ki-67 and S-phase fraction. They suggested that TNC might be a useful marker in selecting patients for adjuvant therapies to reduce the rate of both local and distant cancer recurrences. In a series of 134 operable breast cancer cases, Ioachim et al. [17] investigated the relationships of TNC and found an inverse correlation with estrogen receptor status, a correlation with lymph node status and tumor grade, and an increased mortality risk associated with high levels of TNC expression. A recent trial examined tumor tissue from 86 patients with node-negative breast cancer and found that stromal TNC staining was associated with low relapse-free and overall survival [18]. In a recent study by Helleman et al. [19], high TNC expression was associated with resistance to tamoxifen therapy and shorter distant metastasis-free survival in 139 estrogen receptor-positive and lymph node-positive breast cancer patients. On the other hand, some studies found no correlation between TNC expression and prognosis. For example, Iskaros et al. [20] showed in 115 patients with invasive duct cell carcinoma that although TNC score was significantly positively correlated with high nuclear, histologic, mitotic, and combined grade, there was no correlation between TNC score and long-term survival or with other prognostic factors studied. In another study, Tökés et al. [21] assessed the association of TNC expression, tumor grading, and expression of commonly used histopathologic prognostic factors (p53 and estrogen receptor) in 62 cases of invasive ductal carcinoma of the breast. They found no correlation with prognostic factors p53, Ki-67, and estrogen receptor status. In a small study of 32 Table 5 Multivariate analysis of overall survival

36.1 (0.7) 23.0 (2.1)

97 (3) 51 (10)

Clinical significance of serum tenascin-C levels in breast cancer.

Tenascin-C (TNC) is a key molecule in tissue remodeling, and high levels are observed in many diseases, including heart failure, thrombosis, atheroscl...
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