Decreases in TGF-β1 and PDGF levels are associated with echocardiographic changes during adjuvant radiotherapy for breast cancer

This observational, prospective, single-center study included 73 women with early stage breast cancer or ductal carcinoma in situ (DCIS). All patients received postoperative RT after breast conserving surgery (n = 72) or mastectomy (n = 1), but did not receive chemotherapy. The patient characteristics of the study population are shown in Table 1. The inclusion and exclusion criteria have been previously described [13]. The Tampere University hospital ethics committee approved the study (R10160), and informed consent was obtained from all participants.

The RT protocol has been previously described in detail [14]. Patients received either 50 Gy in 2 Gy fractions or 42.56 Gy in 2.66 Gy fractions. The planning target volume (PTV) was the remaining breast with margins for patients with breast conserving surgery and the chest wall with margins for the post-mastectomy patient. Two patients had positive axillary nodes, and the PTV included axillary and supraclavicular areas.

Serum samples were drawn before RT and on the last day of RT, and they were stored at − 80 °C until analysis. TGF-β1 and PDGF-AB concentrations were determined with an enzyme-linked immunosorbent assay using the reagents from R&D Systems Europe Ltd. (Abingdon, UK). The detection limit and the inter-assay coefficient of variation were 7.8 pg/ml and 5.4% for TGF-b1 and 3.9 pg/ml and 4.6% for PDGF-AB, respectively.

Echocardiographic examinations were performed by a single cardiologist (ST) before and at the end of RT. A commercially available ultrasound machine (Philips iE33 ultrasound system; Philips, Bothell, WA, USA) and a 1–5 MHz matrix-array X5–1 transducer were used to perform the examination, as previously described [13], in a standardized manner following current guidelines [15‐18]. The patients were divided into two groups, one with a ≥ 15% decline and the other with a < 15% decline in tricuspid annular plane systolic excursion (TAPSE), as we earlier reported marked changes in these two parameters [13, 19]. The decline was chosen to represent an approximately 4-mm decrease in TAPSE, which can be considered a clinically meaningful change as in patients with pulmonary hypertension with every 1-mm decrease in TAPSE, risk of death was increased by 17% [20]. Also, in our earlier study the significant average reduction of TAPSE was 2.1 ± 3.2 mm and TAPSE decreased by 4 mm in 39% of patients [19]. Similarly, a ≥ 15% increase and a < 15% increase in the pericardium calibrated integrated backscatter (cIBS) were used to categorize patients into two groups. As the magnitude of a clinically meaningful change is not known for cIBS, a 15% cutoff was used to keep the change similar to the change in TAPSE.

The TGF-β1 and PDGF levels of all 73 patients were measured before and after RT. In these patients, the median (Interquartile Range; IQR) TGF-β1 levels decreased from 25.0 (IQR 21.1–30.3) ng/ml before RT to 23.6 (IQR 19.6–26.8) ng/ml after RT, p = 0.003 (Fig. 1). Similarly, the median PDGF levels decreased from 18.0 (IQR 13.7–22.7) ng/ml before RT to 15.6 (IQR 12.7–19.5) ng/ml after RT, p < 0.001 (Fig. 1). TGF-β1 and PDGF exhibited a strong correlation before RT (Spearman’s rho = 0.802) and after RT (rho = 0.817). The change in TGF-β1 also correlated with the change in PDGF (rho = 0.817) (Fig. 2). There was no significant correlation between change in TGF-β1 or PDGF and the time from surgery to RT (Additional file 1: Table S1) or radiation doses to the heart (Additional file 2: Table S2). Median time from surgery to start of RT was 56.0 (IQR 49.0–64.5) days.

It is generally accepted that TGF-β1 is a pro-fibrotic cytokine that initiates fibrosis in response to RT [1, 2]. Additionally, it plays a role in cardiac remodeling after myocardial infarction [3]. Earlier studies have shown that an increase in TGF-β1 during RT for non-small cell lung cancer is likely to be predictive for the development of radiation pneumonitis [6]. However, some studies do not confirm this finding, and in patients who do not develop radiation pneumonitis, a decrease in TGF-β1 levels is seen [22, 23]. In lung cancer, this decline is thought to represent a decrease in production of TGF-β1 by tumor cells. However, this explanation probably does not explain the decline in TGF-β1 in our study since our patients underwent breast conserving surgery or mastectomy and the likelihood of macroscopic tumor residual is extremely small.

The dynamics of TGF-β1 during adjuvant RT for breast cancer have not been previously reported; however, two studies found that an increased TGF-β1 level before RT was predictive of fibrosis of the breast [4, 5]. Neither of these studies reported the behavior of TGF-β1 during RT. In our patients with a 15% decline in TAPSE, the baseline TGF-β1 level was higher than that in patients without the decline, indicating an association between high TGF-β1 levels and right ventricular dysfunction induced by RT.

TGF-β1 also seems to be a marker of radiosensitivity. A decrease in TGF-β1 levels during RT is associated with a positive response to RT [24]. Additionally, in vitro experiments suggest that blockade of TGF-β1 during RT for non-small cell lung cancer and breast cancer increases radiosensitivity [25, 26]. Therefore, increased TGF-β1 levels seem to be a marker for both radioresistance and radiosensitivity, depending on the tissue in question. As the association of TGF-β1 and echocardiographic has not been studied during RT, we present a novel finding. Levels of TGF-β1 decreased significantly in patients with a decline in TAPSE and an increase in cIBS. TAPSE is in wide clinical use as a reliable measurement of the right ventricular function, and a decline in TAPSE correlates with poor cardiac prognosis in many patient groups [17, 20]. Myocardial reflectivity can be determined with off-line analysis of the echocardiography acquisition (cIBS). Even though the exact basis for the changes in cIBS are not completely understood, an increase in cIBS presents changes in three-dimensional myocardial structure due to factors such as tissue edema or interstitial fibrosis [27].

In studies of cardiac function after an experimental myocardial infarction in mice, blockade of TGF-β1 by an antibody increased mortality and left ventricular dilatation [28]. Another study concluded that early inhibition of TGFβ-1 was detrimental and that later inhibition was beneficial to the cardiac function of mice after an MI, which indicates that the role of TGFβ-1 may be different in various phases of the healing process [29]. In obese, hypertensive patients, an abundance of circulating TGF-β1 is associated with left ventricular filling abnormalities [30]. As concluded by a review conducted by Bujak, the role of TGF-β1 after an MI remains elusive [3].

PDGF consists of two linked chains, designated A, B, C or D. It can be assembled as a hetero- or homodimer [8]. We measured the heterodimer PDGF-AB and found that RT induced a decrease in PDGF that was associated with a decrease in TAPSE and an increase in cIBS. The role of PDGF in long-term adverse effects of RT is not as extensively studied as is the role of TGFβ-1. During RT, PDGF levels were decreased in non-Hodgkin lymphoma patients with varying RT sites, some with preceding chemotherapy and some without [9]. In patients receiving chemotherapy and RT, PDGF and TGF-β1 levels remained unchanged [10]. Because the sites of RT varied, some patients had intact tumors and some patients had chemotherapy that also may influence cytokine levels, indicating that the studies were quite different from our study.

The effect of PDGF has also been studied through inhibition of the PDGF receptor. In mice, treatment with a PDGF receptor tyrosine kinase inhibitor, imatinib, attenuated the development of lung and skin fibrosis [11, 31]. The function of PDGF in cardiac tissue remains elusive, as blockade of PDGF receptor improved cardiac function [32], but injection of exogenous PDGF-AB or PDGF-BB improved heart function after an MI [33‐35].

In our earlier study, we reported changes in TAPSE during RT of left-sided breast cancer patients but found no association with radiation doses [19]. In this study, only patients with available serum samples were included. We found that patients with left-sided breast cancer that had a ≥ 15% decline in TAPSE had higher radiation doses to the whole heart, the left ventricle and the LAD than those with a < 15% decline in TAPSE. There were even more differences in the radiation doses to the whole heart or parts of the heart when patients were grouped according to ≥15% and < 15% increases in cIBS. An increase in cIBS represents an increase in the reflectivity of the cardiac tissue, probably due to structural changes caused by RT-induced inflammation. TAPSE is a parameter depicting longitudinal function of the right ventricle. Its decrease may portray inflammatory changes because the thinness of the right ventricle makes it more sensitive to RT-induced changes [19].

The limitations to our study are that the population is rather small and the follow-up time is very short, as we only studied the changes that occurred during adjuvant RT. At this stage, we do not know if the echocardiographic changes are permanent or if they are associated with the development of fibrosis, which is thought to be responsible for the increased risk of cardiac morbidity after irradiation of the heart [36]. Thus, longer follow-up times are needed to determine whether the behavior of TGF-β1 and PDGF during adjuvant RT depict permanent damage to the heart.