The major findings of this study are the following: 1) In patients with breast cancer there was a relative GLS reduction accompanied by increments in serum NT-proBNP, hs-TnT, VCAM-1 and PICP after 6 months-chemotherapy, without changes in the remaining biomarkers; 2) PICP increment after chemotherapy was more pronounced in the subset of patients who developed subclinical cardiotoxicity; 3) Levels of PICP were directly and independently associated with the relative GLS reduction over the course of chemotherapy; 4) Levels of PICP were inversely associated with LVEF in ACC-HF patients; 5) High levels of PICP at baseline were associated with lack of LVEF improvement after 12 months in ACC-HF patients; and 6) the cardiotoxic agents doxorubicin, cyclophosphamide and trastuzumab stimulated the differentiation of HCFs to a myofibroblast collagen-synthesizing phenotype. To our knowledge, this is the first study evaluating the effects of cancer chemotherapy on myocardial collagen, as assessed non-invasively by using circulating biomarkers associated with histologically-proven MIF. In addition, to the best of our knowledge, this is the first time that the effects of chemotherapy agents on collagen metabolism have been evaluated in HCFs.
It has been widely demonstrated that NT-proBNP and hs-TnT levels increase during cancer therapy, although their utility for cardiotoxicity prediction has been recently questioned.21,30−32 In this study, we have reported chemotherapy-induced increments in these biomarkers associated with a reduction of LV contractility, although the association shown by hs-TnT was not independent of confounding factors. On the other hand, conflicting results have been shown regarding longitudinal changes in circulating VCAM-1, galectin-3 and sST2 during cancer treatment,30,32,33 thus further studies are necessary to confirm the influence of oncologic therapy on the serum levels of these proteins. The current study is the first to report that anthracycline-based-chemotherapy for breast cancer increases the circulating levels of a biomarker that reflects increased collagen deposition in the myocardium,16 being this increase particularly evident in patients developing subclinical cardiotoxicity. In addition, we have found an association between biomarker-assessed myocardial collagen content and a reduction of LV contractility along chemotherapy, independently of relevant clinical factors including chemotherapy cumulative doses and biomarkers of cardiomyocyte damage. Importantly, this association between biomarker-assessed MIF and decreased LV contractility was further confirm in patients with ACC-HF. Even more, longitudinal pilot observations in these patients suggest that the degree of MIF at baseline may influence evolution of LV function over time.
These findings reinforce the notion that, not only the cardiomyocyte injury, but also the presence of MIF seems to be relevant for the impairment of LV contractile function in cancer patients treated with anthracycline-based chemotherapy.7 Mechanistically, these effects combined may result in larger accumulation of collagen encircling cardiac muscle with reduced functionality, further restricting the stretching of muscle fibers and impairing the direct cell-to-cell communication necessary for synchronous activation of cardiomyocytes.34 In this regard, several studies demonstrate a detrimental effect of MIF on the deformation of the LV wall (i.e. reduced GLS), in experimental models35 and in patients with different cardiomyopathies.36–39 Of notice, the PICP levels here observed in breast cancer patients with cardiotoxicity and in patients with ACC-HF were similar to those found in hypertensive heart failure patients with severe MIF.40 It is also important to acknowledge the potential influence of a systemic reparative response after ACC treatment on the levels of serum PICP, with activation of fibrotic processes in organs other than the heart. In this regard, it has been shown that anti-cancer therapies may cause arterial stiffness, with increased collagen production suggested as a potential mechanism, among others, underlying this vascular alteration.41,42 Since arterial stiffness contributes to LVD, we cannot discard that the association found between PICP with LVD in ACC-treated patients is reflecting the combined interaction of cardiac and vascular fibrosis.
Although several studies have reported profibrotic effects of anthracyclines, cyclophosphamide, paclitaxel and trastuzumab in the myocardium of experimental models of cardiotoxicity,8–14 and in rodent cardiac fibroblasts,9,11 the role of these agents in HCFs has not been characterized. In this regard, we describe that doxorubicin, cyclophosphamide and trastuzumab stimulated the differentiation to a myofibroblast collagen-synthesizing phenotype, including enhanced presence of PICP in the extracellular medium. Therefore, we may speculate that cardiotoxic agents used to treat breast cancer may directly stimulate HCFs differentiation and collagen fiber synthesis and deposition. In this regard, it has been suggested that the anthracycline profibrotic effects are mediated through substance P, ROS, STAT3 and atypical G protein Gβ5-related mechanisms.9,11 In addition, cyclophosphamide has been shown to induce human lung fibroblasts premature senescence by activating the MAP kinase signaling pathway.43 Whether these pathways are involved in the pro-fibrotic actions exerted by these agents in HCFs require further studies.
Some limitations of the present study must be acknowledged. First, small sample size may have constrained statistical power to detect differences in the different biomarkers assessed in patients with breast cancer, and preclude from adequately assess the impact of potential confounders in the association and longitudinal analyses in ACC-HF patients. Second, there is lack of follow-up information beyond the considered treatment period in patients with breast cancer, but long-term follow-up is planned and ongoing. Third, serum samples after 1-year follow-up were not available to measure PICP in ACC-HF patients. Fourth, our data cannot be extrapolated to oncologic patients treated with other anti-cancer drugs different from those evaluated in this study. Fifth, because they are descriptive in nature, the associations found between PICP and cardiotoxicity do not establish causality. Finally, we cannot discard extra-cardiac sources of PICP. In this regard, determining the extracellular volume fraction in the myocardium by means of cardiac magnetic resonance could have enriched the description of the influence of myocardial fibrosis on PICP serum levels in ACC-treated patients.
In summary, this study shows for the first time that the circulating biomarker of myocardial collagen type-I deposition, PICP, is elevated in the blood of women receiving breast cancer anthracycline-based chemotherapy. In addition, we report that PICP increment is particularly pronounced in those patients with signs of early LV dysfunction, being the levels of this peptide directly and independently associated with a reduction of LV contractility along chemotherapy. Moreover, we confirm the association of PICP with LV contractile dysfunction in patients with ACC-HF, with high levels of this peptide associated with a lack of improvement in LV contractility over time. Finally, we show for the first time that anthracyclines, cyclophosphamide and trastuzumab exert profibrotic actions, particularly increasing collagen type-I synthesis, in HCFs. These results provide additional evidence for MIF as a cardiotoxic side-effect of anthracycline-based breast cancer treatment, and allow us to speculate that this lesion may be a pathophysiological mechanism underlying LVD in these patients. Finally, these preliminary findings should be considered in the context of a pilot hypothesis-generating study supporting the need of further studies designed to evaluate the prognostic potential of PICP, either by itself or combined with other biomarkers of myocardial remodeling, to monitor cardiotoxicity in larger cohorts of patients under chemotherapy.