Increased circulating resistin levels in early-onset breast cancer patients of normal body mass index correlate with lymph node negative involvement and longer disease free survival: a multi-center POSH cohort serum proteomics study

Approximately one in 300 women aged 40 years or younger is diagnosed with breast cancer in the UK and young age at diagnosis is associated with worse clinical outcomes and greater likelihood of genetic susceptibility (http://​www.​cancerresearchuk​.​org/​health-professional/​cancer-statistics/​statistics-by-cancer-type/​breast-cancer) [1, 2]. Current prognostic biomarkers are based on tumour characteristics, tumour grade and stage, and receptor status. Host factors that may influence prognosis are not currently included in commonly used models [3]. Identifying novel host markers associated with EOBC prognosis may improve our understanding and management of this subgroup of patients.

As a quantitative proteomics approach, the use of chemical labelling with isobaric stable isotope reagents, such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT), has been applied in combination with liquid chromatography–mass spectrometry (LC-MS) techniques for the discovery of candidate cancer biomarkers in serum or plasma [4, 5]. Such methodological approaches provide the distinct advantage of simultaneously measuring protein expression under the same instrumental analysis conditions, thereby reducing experimental bias and improving relative quantitative accuracy and precision [6]. An iTRAQ LC-MS approach that also used a peptide-based affinity enrichment pre-treatment step was applied to plasma samples derived from stage I–III breast cancer patients relative to healthy volunteers [7]. Another iTRAQ LC-MS study that used affinity depletion of the high-abundant proteins was applied to serum samples derived from post-menopausal breast cancer patients relative to healthy controls [8]. In this study, however, we utilized quantitative LC-MS proteomic methods that do not depend on prior affinity enrichment or depletion of plasma/serum which may compromise their analysis for clinically relevant protein markers [5, 9]. In this capacity, the entire serum protein content was subjected to quantitative proteomic analysis. Using serum from a cohort study of early-onset breast cancer cases, we explored the potential for quantitative discovery proteomics to reveal novel markers of poor outcome in young women with EOBC [2].

Improvements made in breast cancer survival have been associated with the wider use of neo/adjuvant chemotherapy such as anthracycline/taxane-based treatment [37]. Routine immunohistochemical analysis is used for both prognosis and predictive markers of response to hormonal therapy and trastuzumab (ER/PR and HER2 respectively). Young age [38, 39] and obesity [2] at breast cancer diagnosis have been reported to be independent prognostic markers of adverse disease outcome. The aim of this study was to find serum proteomic markers of additional prognostic relevance to EOBC outcomes.

This study implemented a high-precision quantitative serum proteomics discovery analysis followed by targeted serum ELISA-based validation in an independent sample set of non-obese EOBC patient samples (Fig. 1). The applied proteomics method achieved the highest degree of proteome coverage in breast cancer serum to date (5346 unique proteins with peptide FDR p ≤ 0.05). The methodological feature that led to this comprehensive proteome result was its ability to analyse non-depleted serum that also contains exosome-enriched and other extracellular vessicle- derived proteins in addition to directly secreted proteins, as reported previously [9, 12, 14]. Such an in-depth analysis was deemed essential for the unbiased interrogation of expected systemic effects and their affiliated biological pathways and networks induced by treatment.

Hierarchical clustering analysis of all 812 differentially expressed proteins (DEPs) is presented in heatmap format in Fig. 2a. The DEPs were then subjected to canonical pathway analysis, which achieved significant enrichment for the insulin signalling (p = 0.015) (Fig. 2b) and glycolysis/gluconeogenesis (p = 0.011) pathways (Fig. 2c). Interestingly, the majority of observed proteins that encoded for both of these pathways were of exosomal origin, as listed in the manually curated ExoCarta Web-based compendium (http://​www.​exocarta.​org) [40‐42]. Of relevance, all enzymes mapping to the glycolysis/gluconeogenesis pathway were upregulated in the poor outcome group, suggesting that poor-prognosis patients catabolize glucose more actively compared to patients with longer survival (Fig. 2c). One noteworthy enzyme found to be upregulated in the poor outcome group was the pyruvate kinase M2 isoform (PKM2) known to play an important role in tumorigenesis. As observed in different types of cancers, including breast cancer, pyruvate kinase expression shifts to the PKM2 isoform in order to utilize glucose more efficiently to generate biomass under anaerobic conditions [43]. The functional involvement of the insulin signalling and the glycolysis/gluconeogenesis pathways were further verified with Ingenuity Pathway Analysis that showed significant enrichment for glucose and fatty acid metabolism (Fig. 2d) and included resistin, a secreted protein, as one of its key nodal components. We focused on serum resistin given its association with the insulin signalling and glycolysis/gluconeogenesis pathways as a candidate marker of EOBC prognosis.

In agreement with the discovery cohort (Fig. 3a), resistin was found to be upregulated in the good outcome group in the normal weight validation cohort (Fig. 3b). To address accurate protein inference, ELISA was used as the measurement approach for the validation cohort because it allowed the analysis of the intact form of resistin, whereas bottom-up proteomics, as used in this study, allows the assessment of protein expression at the derived peptide level resulting from the trypsin proteolysis step.

In this work, both linear and generalized linear regression analysis confirmed that ER, PR and HER2 exhibited a significant degree of interdependence (p < 0.05) (Additional file 6: Section 6). A receiver operating characteristic (ROC) curve (Fig. 4a) and associated cost curve (Fig. 4b) were used to assess the value of resistin in outcome prediction between the two groups in this study, The AUC measure of the ROC curve indicated a moderate level of success for utilizing resistin measures to predict outcome. Using the measure of true positives, true negatives, false positives and false negatives (Fig. 4c), serum resistin provided an accuracy of 0.652, a sensitivity of 0.667 and a specificity of 0.637. We explored resistin expression at the tissue level using an in-silico meta-analysis micro-array database, the Kaplan–Meier Plotter software tool (http://​kmplot.​com/​analysis/​). Consistent with the serum observations in our current study, this analysis showed that high tissue levels of resistin were associated with longer disease-free survival (p < 0.001) (Fig. 4d).

Resistin is a pro-inflammatory molecular that has been implicated in obesity-mediated type 2 diabetes. Obesity is a host factor that adversely influences breast cancer prognosis [2, 42]. There is evidence that insulin resistance may develop after breast cancer adjuvant therapy [41], and a recent prospective study reported that increased resistin levels coincided with the concurrent increase in serum insulin and insulin resistance following treatment (surgery followed by chemotherapy and radiotherapy) among stage II–III breast cancer patients in an adiposity-independent way [35]. It is therefore possible that derangement of glucose metabolism through insulin resistance may be a result of late toxic effects of chemotherapy possibly due to impaired pancreatic beta-cell function. However, in our present study all patients received chemotherapy and so any differential effect cannot be due to the chemotherapy alone. Recent reports strongly suggest that resistin production in humans is largely from macrophages rather than adipose tissue alone (also known to contain macrophages) [30, 33, 44]. Insulin pathophysiology has been associated with inflammatory markers independent of BMI in subjects at risk of type 2 diabetes [45]. Additionally, in transgenic mice, production of human resistin from macrophages was associated with increased inflammation and contributed to the acquisition of insulin resistance [33]. Our current proteomic findings add to the evidence suggesting that resistin is a potential surrogate marker of disturbed insulin pathophysiology and inflammation that could provide an explanation for the observed association between higher resistin level and improved DFS.

As an ancillary finding, resistin levels were significantly higher in LN-positive vs LN-negative patients, irrespective of outcome group (p = 0.0037) (Fig. 3c). A regression model further examined this trend where the LN status demonstrated a significant association with resistin measurements. Resistin overexpression was found to correlate with node-negative status (p = 0.0428). This trend, in combination with the results from the association testing, provide further evidence that resistin and nodal status could be linked (Additional file 6: Section 6). During inflammation, macrophages can be both a major source of resistin and themselves able to respond to resistin in an autocrine loop, leading to an increase in pro-inflammatory ‘M1-like’ macrophages and a reduction in anti-inflammatory ‘M2-like’ macrophages [33, 46]. Given that the lymph node status existed at presentation and all patients received chemotherapy, we considered whether the overexpression of resistin per se may have influenced the tumour micro-environment to exert a suppressive effect on tumour cell motility or extravasation. The association of anti-inflammatory ‘M2-like’ monocytes and macrophages with metastases in preclinical models [47] provides a possible mechanism whereby increased resistin levels could lead to a lower potential for metastatic spread by promoting a pre-existing pro-inflammatory tumour microenvironment. To further explore this hypothesis, the post-priori examination of the downstream differentially expressed proteins between the good vs poor outcome groups using the Diseases & Functions module of Ingenuity Pathway Analysis identified the inflammatory response, leucocyte infiltration (also implicating resistin), lymphocyte migration and recruitment of phagocytes to be significantly induced biological processes (p < 0.0001, z-score > 2) (Fig. 5). Overall, improved prognosis associated with increased resistin levels may indicate an immunomodulatory role of this protein during early breast tumour development limiting the ability of the tumour primary cells to spread to distant sites. Further examining the mechanistic link between circulating resistin levels and patient LN status was beyond the scope of the present study; future studies will be required to explore this hypothesis. This is a relatively small study, and a larger follow-up study is warranted, ideally with pre-treatment serum samples to determine whether the observed specific correlation with metastasis to axillary lymph nodes holds true at all ages. A potential technical limitation was the sample pooling strategy used in the discovery phase, which did not permit the assessment of anticipated inter-individual heterogeneity in protein expression levels. However, extensive sample pooling is more likely to find larger, more consistent, protein differences that are therefore more likely to replicate. In addition, the accuracy of relative protein quantitation for resistin was validated with ELISA measurements against individual serum specimens from a separate validation cohort, and from the in-silico analysis of an independent cohort at the tissue level.

A high-precision serum proteomics-based pipeline identified increased serum resistin to positively correlate with disease-free survival independent of BMI in women with EOBC. High resistin levels were associated with less axillary lymph node involvement at presentation and better survival. We hypothesize that individuals with early breast cancer who have relatively higher resistin levels may provide an environment from which tumours are less likely to metastasize. Further prospective studies are needed to confirm these findings and elucidate the mechanistic role of resistin in EOBC patients.