Abstract
Introduction: Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a newly discovered tumor suppressor gene. It contains one epidermal growth factor (EGF)-like domain. Many studies have shown that proteins contain (EGF)-like domain structure affect a variety of biological actions. However, little is known about the relationships between NGX6 expression and biological behaviors in invasive ductal breast carcinoma (IDBC). The study discussed the association of ultrasonographic features with NGX6 expression and prognosis in IDBC. Methods: Ultrasonographic feature and clinical data in 122 patients with IDBC were retrospectively analyzed. NGX6 expression of IDBC was measured using immunohistochemistry methods. Results: The incidence of the burr sign, lymph node metastasis and abundance blood flow in NGX6 expression negative groups were higher than those in positive groups. Kaplan-Meier analysis showed that the association between NGX6 positive expression and higher disease-free survival (DFS) or higher overall survival (OS); Lymph node metastasis is associated with lower DFS or lower OS; Lower blood flow grade is associated with higher DFS. In univariate and multivariate survival analysis, NGX6 expression, lymph node metastasis, TNM and the blood flow grade were the independent prognostic factors for DFS and OS of IDBC. Conclusions: ultrasonographic features are associated with NGX6 expression in IDBC. NGX6 is involved in the invasion and metastasis activity of IDBC. Our results suggest that NGX6 may be employed as a promising prognostic factor and useful therapeutic target for IDBC. Combination of ultrasonic findings and NGX6 detection may yield clinically useful information for IDBC prognosis.
Keywords: Invasive ductal breast carcinoma (IDBC), ultrasonography, NGX6, prognosis
Introduction
Breast cancer is the most prevalent cancer among women. The mortality of breast cancer continually rose over the last few years. Ultrasound is one of the most effective tumor diagnostic techniques. Biological characteristics of tumor such as growth, invasion and metastasis can be observed, non-invasively and accurately, using ultrasonic technique [1-3]. NGX6 is a newly discovered tumor suppressor gene (GenBank accession no. AF188239). NGX6 gene contains one epidermal growth factor (EGF)-like domain, and that is one of the most important biological characteristics. Many studies have shown that proteins contain (EGF)-like domain structure affect a variety of biological actions, such as tumor growth, invasion and metastasis [4-7]. However, little is known about the relationships between NGX6 expression and biological behaviors in breast cancer. The study discussed the association of ultrasonographic features with expression of NGX6 proteins and prognosis in IDBC in order to provide valuable information for clinical prognosis.
Materials and methods
Patients
This study was approved by the Ethics Committee of Third Xiangya Hospital. From January 2009 to April 2011, 130 patients scheduled for surgery took preoperative ultrasound and were confirmed to be IDBC with pathological examination. 8 women who were pregnant or nursing were also excluded. 122 patients presented with single tumor were included in the study. All patients were female with a mean age 51.8 years ranging from 27 to 76 years. Data on the patients’ medical history, tumor features, histologic classification and treatment modalities were recorded. All of the patients undergoing breast surgery received post-operative radiotherapy. Adjuvant chemotherapy was administered depending on the risk of recurrence in accordance with the National Comprehensive Cancer Network guidelines [8]. Dates and causes of death were obtained from the medical records. Survival assessment was last performed in March 2014. All the subjects signed the informed consent.
Ultrasound examination
Conventional ultrasound imaging was performed in patients one week before surgery with using a scanner (Siemens, ACUSON, S2000, Germany) with a multi-frequency linear 9 MHz transducer. Breast scanning was in clockwise direction along the center of the nipple. The content observed included the morphology of mass, size, border, internal echo, internal microcalcifications, axillary lymph node and blood supply. Blood flow signal was graded by Adler classification method [9].
Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded tissue sections (4 μm in thickness) were deparaffinized and subsequently hydrated with an ethanol gradient. Antigen retrieval was performed in a 10 mmol/L sodium citrate (pH 6.0) for 5 min with a high pressure. The tissue sections were immersed in 3% H2O2 for 10 min to inactivate endogenous peroxidase. 10% goat serum was added to the tissue sections and incubated for 30 min at 37°C. The sections were incubated with Rabbit anti-NGX6 monoclonal antibodies (1:200 dilution, Abcam, USA) overnight at 4°C, and then incubated at 37°C for 30 min with a secondary antibody against rabbit and mouse immunoglobulins (EnVision, DAKO, Denmark). Afterwards, the sections were stained with DAB for 5 min. The cytoplasm and cell membrane stained in yellow brown were the positive pattern. Five areas representative were randomly observed in the perspective of 400 times microscope. The number of positive cells counts was not less than 200 in each observed area. Classification is done according to the strength of cells staining and the proportion of the positive cell. The number of positive cells < 10% was defined as negative, and 10% or higher was defined as positive [10]. All immunohistochemical analyses were evaluated by two pathologists blindly and independently.
Statistical analysis
The data was processed by SPSS13.0 statistical analysis package, the measurement data used variance test, counting information using chi-square test. The Kaplan-Meier method was used to estimate the survival outcomes and groups were compared using the log-rank test. The Cox proportional hazards model was used for multivariate analysis. The significance level was set at P < 0.05 (both sides).
Results
Ultrasonographic features in patients with IDBC
Among 122 patients with IDBC, there were 68 (55.7%) cases with the tumor diameter greater than 2.0 cm and 54 (44.3%) cases with diameter less than 2.0 cm; 76 (62.3%) cases with mass around a burr (Figure 1A) and 74 (60.7%) cases with mass with microcalcifications (Figure 1B). Blood flow signal in tumors were graded with following: I-II level in 45 (36.9%) cases (Figure 1C), III level in 77 (63.1%) cases (Figure 1D) lymph node metastasis in 69 (56.6%) cases.
Figure 1.
Ultrasonographic features in patients with IDBC. A. Edge burr sign in IDBC. B. Microcalcification in IDBC. C. Blood flow I-II grade in IDBC. D. Blood flow III grade in IDBC.
NGX6 expression in IDBC tissue
Among 122 cases of IDBC, positive expression was found in 55 (45.1%) cases (Figure 2A) and negative expression was found in 67 (54.9%) cases (Figure 2B).
Figure 2.
Representative immunohistochemical staining of NGX6 expression in IDBC tissues. A. Positive expression of NGX6; B. Negative expression of NGX6. Representative images are shown at × 400 magnifications.
Relationship between Ultrasonographic features and NGX6 expression in IDBC
Mass edge burr sign, blood flow grade and lymph node metastasis in IDBC were associated with NGX6 expression. Tumor size and microcalcification were not associated with NGX6 expression, as shown in Table 1.
Table 1.
The relationship between Ultrasonographic features and NGX6 expression in IDBC
| Ultrasonographic features | n | NGX6 | χ2 | P | |
|---|---|---|---|---|---|
|
| |||||
| (+) | (-) | ||||
| Tumor size | |||||
| ≥ 2 cm | 68 | 31 | 37 | 0.016 | 0.900 |
| < 2 cm | 54 | 24 | 30 | ||
| Edge burr sign | |||||
| Present | 76 | 27 | 49 | 7.434 | 0.006 |
| Absent | 46 | 28 | 18 | ||
| Microcalcification | |||||
| Present | 74 | 33 | 41 | 0.018 | 0.893 |
| Absent | 48 | 22 | 26 | ||
| Lymph node metastasis | |||||
| Present | 69 | 25 | 44 | 5.025 | 0.025 |
| Absent | 53 | 30 | 23 | ||
| Blood flow grade | |||||
| I-II | 45 | 26 | 19 | 4.642 | 0.031 |
| III | 77 | 29 | 48 | ||
Survival analysis
In the Table 2, univariate Cox regression analysis revealed that lymph nodal status, TNM stage, blood flow grade and NGX6 expression were significantly associated with the DFS and OS.
Table 2.
Univariate Cox regression analysis of potential prognostic parameters associated with DFS and OS in breast cancer patients
| Variable | DFS | OS | ||
|---|---|---|---|---|
|
|
|
|||
| HR (95% CI) | P value | HR (95% CI) | P | |
| Ages, years | ||||
| ≥ 35 years vs. < 35 years | 0.734 (0.244-1.369) | 0.235 | 0.858 (0.436-1.653) | 0.421 |
| Family history | ||||
| Yes vs. No | 0.838 (0.452-1.495) | 0.476 | 0.941 (0.658-1.254) | 0.256 |
| Menopause status | ||||
| Pro vs. Pre | 0.987 (0.364-1.883) | 0.172 | 1.575 (1.269-1.786) | 0.195 |
| Tumor size | ||||
| ≥ 2 cm vs. < 2 cm | 2.144 (1.234-3.389) | 0.127 | 1.867 (0.988-3.742) | 0.248 |
| Edge burr sign | ||||
| Present vs. absent | 1.035 (0.934-1.367) | 0.295 | 5.864 (2.986-8.467) | 0.342 |
| Microcalcification | ||||
| Present vs. absent | 3.547 (1.838-5.374) | 0.176 | 4.859 (2.743-6.789) | 0.273 |
| Lmph node metastasis | ||||
| Present vs. absent | 4.562 (2.258-7.355) | 0.012 | 6.538 (3.987-11.563) | 0.005 |
| Blood flow grade | ||||
| I-II vs.III | 1.735 (1.234-2.361) | 0.033 | 3.436 (2.658-4.237) | 0.015 |
| ER status | ||||
| Positive vs. Negative | 1.170 (0.725-1.845) | 0.520 | 1.236 (0.748-1.554) | 0.132 |
| PR status | ||||
| Positive vs. Negative | 0.717 (0.439-1.173) | 0.232 | 2.867 (1.752-3.789) | 0.432 |
| HER2 status | ||||
| Positive vs. Negative | 1.934 (1.072-3.236) | 0.084 | 2.909 (1.253-3.745) | 0.106 |
| NGX6 status | ||||
| Positive vs. Negative | 2.531 (1.832-3.347) | 0.002 | 1.653 (1.257-2.172) | 0.008 |
| Adjuvant chemotherapy | ||||
| Yes vs. No | 1.494 (0.554-2.471) | 0.166 | 2.867 (1.732-3.789) | 0.093 |
| TNM stage | ||||
| I-II vs. III | 2.489 (1.597-3.275) | 0.018 | 8.985 (7.773-12.075) | 0.023 |
Kaplan-Meier analysis of DFS and OS in a subgroup of IDBC, according to the status of NGX6 expression, Lymph node metastasis and blood flow grade, and Kaplan-Meier curves of survival are shown below. This reveals the association between NGX6 positive expression and higher DFS (P = 0.023, log rank test) or higher OS (P = 0.018, log rank test) (Figure 3).
Figure 3.
Kaplan-Meier analysis of DFS, OS in patients with IDBC, according to the status of NGX6 expression (positive or negative). P-value obtained from log-rank test, n = 122. It’s showing the association between of NGX6 positive expression and higher DFS or higher OS in patients with IDBC. NGX6 (+): NGX6 expression positive group; NGX6 (-): NGX6 expression negative group.
Lymph node metastasis is associated with lower DFS (P = 0.045, log rank test) and lower OS (P = 0.026, log rank test) (Figure 4).
Figure 4.
Kaplan-Meier analysis of DFS, OS in patients with IDBC, according to the status of lymph node metastasis (positive or negative). P-value obtained from log-rank test, n = 122. It’s showing the association between of lymph node metastasis is associated with lower DFS and lower OS in patients with IDBC. LN metastasis (+): Lymph node metastasis positive group; LN metastasis (-): Lymph node metastasis negative group.
Lower blood flow grade is associated with higher DFS (P = 0.035, log rank test). However, no significant association has been identified in relation to OS (P = 0.093, log rank test) (Figure 5).
Figure 5.
Kaplan-Meier analysis of DFS, OS in patients with IDBC, according to the status of blood flow grade (I-II or III) as determined by ultrasonography. P-value obtained from log-rank test, n = 122. It’s showing the association between of lower blood flow grade and higher DFS in patients with IDBC.
Multivariate Cox regression analysis of DFS and OS in breast cancer patients, NGX6 expression, lymph node metastasis, TNM and blood flow grade was the independent prognostic factors for DFS. NGX6 expression, Lymph node metastasis and TNM were the independent prognostic factors for OS. As shown in Table 3.
Table 3.
Multivariate Cox regression analysis of DFS and OS in IDBC patients
| Survival | Influencing factors | HR (95% CI) | P |
|---|---|---|---|
| DFS | Tumor size | 2.465 (1.176-3.708) | 0.324 |
| Lmph node metastasis | 3.548 (1.235-5.984) | 0.035 | |
| Blood flow grade | 2.217 (1.368-3.138) | 0.023 | |
| HER2 status | 3.354 (2.252-4.258) | 0.079 | |
| NGX6 status | 1.948 (1.327-2.636) | 0.025 | |
| TNM stage | 3.469 (1.254-5.578) | 0.032 | |
| OS | Tumor size | 0.852 (0.454-1.326) | 0.224 |
| Lmph node metastasis | 5.487 (2.495-9.328) | 0.047 | |
| Blood flow grade | 3.543 (1.258-6.084) | 0.224 | |
| HER2 status | 2.514 (1.289-4.038) | 0.068 | |
| NGX6 status | 2.318 (1.752-2.983) | 0.015 | |
| TNM stage | 6.568 (3.693-9.165) | 0.043 |
Discussion
The molecular basis of tumor determines biologic behavior, tissue pathological morphology and imaging changes. Many literatures confirmed that there are correlations between tumor imaging and prediction factors, including relevant genetic expression, clinicopathological features [11-15]. Many information obtained from these types of studies contributed to treatment selection and prognostic estimation for a variety of tumors. NGX6 is a newly identified tumor suppressor gene. NGX6 expression level can reflect the progress of tumor invasion and metastasis level [4-8,16-19]. Combination of ultrasonic findings and NGX6 detection in breast cancer may yield clinically useful information.
In this study, occurrence rate of edge burr, lymph node metastasis in NGX6 negative expression group was higher than NGX6 positive expression group in IDBC. Pathological basis of edge burrs presented as tumor infiltration and promotion to the surrounding tissues [20,21]. This data showed that NGX6 gene expression was correlated with edge burr and lymph node metastasis. NGX6 gene is likely involved in the invasion and metastasis activity of IDBC.
Color Doppler flow imaging (CDFI) observation showed that the blood flow of breast cancer was richer in the NGX6 express negative group than in the NGX6 positive group. Tumor growth and metastasis are inseparable from the formation of blood vessels. Tumor vessels enlarge and actively engage in sprouting and branching to meet the metabolic demands of adjacent malignancy [22]. NGX6 could influence many angiogenic molecules, such as up-regulating p19 and down-regulating EphB4 [23]. Lian P et al [24] found microvessel density (MVD) of NGX6 transfection tumor model was significantly lower than that of non-transfection tumor model. These suggest that NGX6 expression is associated with the tumor vascular abundance and NGX6 gene may participate in the process of inhibiting angiogenesis.
In univariate and multivariate survival analysis of breast cancer patients, the data showed that DFS and OS are significantly associated with NGX6 expression, lymph node metastasis and TNM. To the best of our knowledge, this is the first study examining the relationship between NGX6 and ultrasonographic features and prognosis in patients with breast cancer. NGX6 expression was demonstrated to be independently associated with ultrasonographic imaging features and clinical outcomes, highlighting that NGX6 may be employed as promising prognostic factor and useful therapeutic targets to improve the survival of breast cancer patients.
It is well known that TNM and lymph node metastasis are prognostic factors for breast cancer and is responsible, at least in part, for the mortality from breast cancer in women. Our data were consistent with these reports [25-29]. Blood flow grade status was a significant predictor of DFS. However, it was not identified as prognostic factor affecting OS. Many other factors including tumor size, ER, PR and HER2 status were also not identified as prognostic factor in our data statistical analysis. One of the reasons is that only a small number of patients were examined in this study, making it difficult to reach statistical significance. We hope to study a greater number of patients in the future.
In conclusion, ultrasonographic features are associated with NGX6 expression in IDBC. NGX6 gene expression is likely involved in the invasion and metastasis activity. NGX6 expression, Lymph node metastasis, TNM and blood flow grade are the important independent prognostic factors for IDBC. NGX6 detection and ultrasound findings may complement each other and provide important prognosis information for breast cancer patients.
Acknowledgements
This work was supported by grants from the Science Foundation of Hunan Province (2013FJ4094), The Medical Foundation of Hunan Province (B2013-023), High-level medical project (225) and the Educational Project of Hunan province (XJK013CGD081) China.
Disclosure of conflict of interest
None.
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