Prognostic and predictive impact of stroma cells defined by PDGFRb expression in early breast cancer: results from the randomized SweBCG91RT trial

Purpose Predictive biomarkers are needed to aid the individualization of radiotherapy (RT) in breast cancer. Cancer-associated fibroblasts have been implicated in tumor radioresistance and can be identified by platelet-derived growth factor receptor-beta (PDGFRb). This study aims to analyze how PDGFRb expression affects RT benefit in a large randomized RT trial. Methods PDGFRb was assessed by immunohistochemistry on tissue microarrays from 989 tumors of the SweBCG91RT trial, which enrolled lymph node-negative, stage I/IIA breast cancer patients randomized to RT after breast-conserving surgery. Outcomes were analyzed at 10 years for ipsilateral breast tumor recurrence (IBTR) and any recurrence and 15 years for breast cancer specific death (BCSD). Results PDGFRb expression correlated with estrogen receptor negativity and younger age. An increased risk for any recurrence was noted in univariable analysis for the medium (HR 1.58, CI 95% 1.11–2.23, p = 0.011) or PDGFRb high group (1.49, 1.06–2.10, p = 0.021) compared to the low group. No differences in IBTR or BCSD risk were detected. RT benefit regarding IBTR risk was significant in the PDGFRb low (0.29, 0.12–0.67, p = 0.004) and medium (0.31, 0.16–0.59, p < 0.001) groups but not the PDGFRb high group (0.64, 0.36–1.11, p = 0.110) in multivariable analysis. Likewise, risk reduction for any recurrence was less pronounced in the PDGFRb high group. No significant interaction between RT and PDGFRb-score could be detected. Conclusion A higher PDGFRb-score conferred an increased risk of any recurrence, which partly can be explained by its association with estrogen receptor negativity and young age. Reduced RT benefit was noted among patients with high PDGFRb, however without significant interaction.


Introduction
Radiotherapy (RT) in combination with breast conserving surgery (BCS) is currently the preferred treatment over mastectomy for patients with early stage breast cancer. Nevertheless, a minority of these patients will suffer from local Axel Stenmark Tullberg and Reidunn Jetne Edelmann contributed equally as second authors and also Erik Holmberg and Arne Östman contributed equally as second last authors. recurrences during the first decade after surgery [1,2]. Classic histopathological variables are unable to identify patients with different proportional benefits from adjuvant RT [2]. An increasing focus is being put on the microenvironment as a modulator of the benefit from adjuvant RT. Recently, a high number of tumor-infiltrating lymphocytes was shown to independently reduce the recurrence risk of early stage breast cancer patients within a randomized RT trial. Consequently, patients with low levels of tumor-infiltrating lymphocytes may represent a subgroup with an increased RT benefit [3]. Preclinical studies have indicated that stroma cells can modulate radiosensitivity of tumor cells [4][5][6][7], but non-leukocytic stroma cells have not yet been explored as potential predictive markers for benefit of RT in invasive breast cancer.
Platelet-derived growth factor receptor beta (PDGFRb) is a key regulator of fibroblasts, pericytes and smooth muscle cells (reviewed in [8][9][10]). The role of stromal PDGFRb expression in progression and treatment response of invasive breast cancer is still not fully understood. A high expression of PDGFRb in the tumor stroma has been associated with unfavorable clinicopathological variables and shorter recurrence free and breast cancer specific survival, univariably, in a population-based cohort [11] although there are also studies which have failed to confirm the prognostic effect [12]. However, the application of a gene expression signature reflecting PDGFRb-activation stably indicated prognostic relevance of a high signature score for shorter recurrence free survival and/or breast cancer specific survival in four independent patient cohorts [13].
Stromal PDGFRb expression could also be treatment predictive. Higher expression of PDGFRb has been associated with a significantly decreased benefit from tamoxifen in ER-positive invasive breast cancer [14]. The mechanistic relationship has not yet been elucidated.
Thus, findings from these studies underline the importance of a study design that can discriminate prognostic and treatment related effects. Furthermore, these observations also point towards the complex interplay between PDGFRb as a potential marker of distinct stroma cell populations and at the same time as an active signaling receptor driving tumor progression.
Preclinical models of different solid tumor types have suggested potential mechanisms of how PDGF-activated stroma cells can modulate treatment effects and prognosis (reviewed in [9]). These mechanisms range from modulation of interstitial fluid pressure impairing drug uptake [15][16][17] to induction of a basal like tumor cell and promotion of dissemination through paracrine signaling [18][19][20]. With regards to RT, experimental models have provided evidence for general radioprotective effects of fibroblasts on cancer cells [21][22][23], but a role of PDGF signaling in these models has not yet been demonstrated.
As the current literature is conflicting regarding the function of stromal PDGFRb on prognosis as well as treatment response in invasive breast cancer, the purpose of the present study was to analyze the prognostic and predictive impact of stromal PDGFRb on ipsilateral breast tumor recurrence (IBTR), any recurrence and breast cancer specific death (BCSD) in a large and clinically well-annotated randomized RT trial of early stage breast cancer patients.

Patient cohort
The retrospective analysis included patients from the SweB-CG91RT trial who have been described elsewhere [24,25] (Table 1). In short, 1178 lymph node negative (N0) patients with stage I or IIA breast cancer were randomly assigned to BCS with or without whole breast RT between the years 1991 and 1997 and followed for a median time of 15.2 years (Fig. 1). Tumor blocks from the initial surgery were retrieved, and tumors were classified according to the St Gallen International Breast Cancer Conference Expert Panel 2013 using immunohistochemical panels.
ER and HER2 evaluation has been described previously [25]. In brief, the cutoff used to consider a tumor ER positive was 1%, for PgR the cutoff was ≥ 20% to distinguish luminal A-like from luminal B-like tumors. Triple negative tumors were defined as negative for ER, PgR and HER2. HER2 was considered positive if 3+ on immunohistochemistry level or amplified on silver in situ hybridization [25]. Patients were well balanced regarding clinicopathological baseline characteristics across the treatment arms as shown previously [24].

Immunohistochemistry (IHC)
The Ventana Benchmark Discovery autostainer system (NexES V10.6) was used for immunohistochemical staining of PDGFRb on 4 μm freshly cut sections from formalinfixed paraffin embedded tissue microarray (TMA) blocks. The protocol included extended antigen retrieval with pH10 Tris buffer (Sigma-Aldrich and Merck Kgaa, Darmstadt, Germany) and incubation for 1 h at 37 °C with the primary antibody (rabbit monoclonal anti-PDGFRb antibody, clone 28E1, #3169 Cell Signaling, Danvers MA, US) diluted at 1:100 dilution in Discovery Antibody Diluent (Ventana, Tuscon, Arizona, US). Chromogenic detection was performed using the Discovery OmniMap anti-rabbit HRP (RUO) kit (Ventana) with secondary antibody incubation for 32 min at room temperature. Hematoxylin II was applied for 10 min and subsequent bluing for 6 min (Ventana) in order to obtain counterstaining. Antibody-based cross detection of the structurally related PDGFRa was excluded as described previously [26].

Statistics
Time to IBTR as first event within 10 years was used as primary endpoint. Secondary endpoints were time to any breast cancer recurrence within 10 years (IBTR, regional recurrence or distant recurrence) and time to breast cancer specific death (BCSD) within 15 years. Regional recurrence, distant recurrence and death were considered competing risks for IBTR.
Known clinical variables were tested first in univariable and then, if significant, in multivariable analysis including age group, histological grade, subtype and RT treatment. Subtype was kept in multivariable analysis, despite not being significant in univariable analysis, because of the biologic relevance. Hazard ratios (HRs) were calculated with causespecific Cox proportional hazards regression to reflect the biologic effect of RT in the presence of competing risks. Correlation analysis between clinicopathologic parameters and stromal PDGFRb status was tested using Spearman's Rank test.
Figures of cumulative incidence were created according to the method by Fine and Gray [28]. p values for the hazard ratio between compared groups were denoted P CIF in the plots. p values < 0.05 were considered significant. STATA The proportional hazards assumption was checked graphically and tested with Schoenfeldt's test. It was violated for RT, histological grade, subtype and RT: PDGFRb score and these values should thus be interpreted as the mean value over 10 years.

Marker evaluation
Out of 1004 cases included in the TMA, 989 cases were successfully scored (Figs. 1, 2). Using Cohen's kappa statistics, the inter-rater agreement was in the moderate range for scoring of the average staining intensity (κ = 0.59) and of the positive stroma fraction (κ = 0.45).

Prognostic potential of stromal PDGFRb expression
No prognostic impact was observed for any of the PDG-FRb score groups with regards to IBTR at 10 years after BCS (Fig. 4a, Table 2). For any recurrence, a significantly increased risk was detected in univariable analysis for patients with a medium (HR 1.58, CI 95% 1.11-2.23, p = 0.011) or high PDGFRb score (HR 1.49, CI 95% 1.06-2.10, p = 0.021) as compared to the PDGFRb low score group (Fig. 4b, Table 2). In a multivariable analysis including histological grade, age, RT and subtype, the significance remained for the PDGFRb medium (HR 1.46, CI 95% 1.01-2.11, p = 0.042) but not the PDGFRb high score group (HR 1.32, CI 95% 0.93-1.88, p = 0.125) ( Table 2). PDGFRb score was not significantly associated with risk of BCSD within 15 years from diagnosis (Fig. 4c, Table 2).  (Fig. 5a, Table 3).  No significant interaction between RT and PDGFRb score could however be detected for IBTR (p = 0.153) or any recurrence (p = 0.320) (Fig. 5b, Table 3). No benefit from RT regarding BCSD was observed for any of the PDGFRb score groups at 15 years after breast conserving surgery and no significant interaction between PDGFRb score and RT was noted for BCSD (p = 0.636) (Fig. 5c,  Table 3).

Discussion
Our study suggests that patients with higher expression of PDGFRb might have an increased risk of any breast cancer recurrence, but due to correlation with younger age and ER negativity, a function of PDGFRb as independent prognostic marker could not be demonstrated. Furthermore, our analyses demonstrated, both univariably as well as multivariably, that patients of the high PDGFRb score group derive less benefit from adjuvant RT in terms of IBTR as compared to the low and medium score groups. However, since the interaction test between PDGFRb and RT was not significant, our data does not confirm stromal PDGFRb expression as a predictive biomarker for RT benefit in early stage invasive breast cancer. PDGFRb is a key regulator of fibroblasts and mural cells and has been previously suggested, both by functional and correlative studies, to play a role in the progression and treatment response of invasive breast cancer [8,9,11,14,29]. However, published findings are partly conflicting most likely due to study designs not allowing a clear discrimination of prognostic and treatment related effects. In this study we analyzed the prognostic and predictive impact of stromal PDGFRb in the randomized SweBCG91RT trial.
PDGFRb has previously been shown to correlate with unfavorable clinicopathological variables such as ER negativity, younger age and higher histological grade [11,14]. These associations were confirmed in our study and could explain part of the prognostic effect of PDGFRb expression. However, the prognostic influence remained significant in multivariable analysis regarding any recurrence for the PDGFRb medium score group patients, which indicates that PDGFRb can provide independent prognostic information. In the present study, a tendency towards higher IBTR risk among patients with higher PDGFRb expression was also noted, although these results were not significant. These results are in line with previous reports describing a similar association between high stromal PDGFRb expression and shorter time to recurrence in a population-based cohort including both patients with negative and positive nodal status as well as patients with and without adjuvant endocrine treatment, chemotherapy or the combination [11].
PDGFRb is mainly expressed on fibroblasts and vascular mural cells, and both stromal cell types are a key source of growth factors and cytokines [9,10]. The secretome of cancer associated fibroblasts (CAFs) has been connected to therapy resistance in breast cancer [19,[30][31][32][33]. In addition, CAFs can affect other cells of the tumor microenvironment such as immune cells and vascular cells and thereby indirectly influence tumor progression and therapy efficacy (reviewed in [34][35][36]). CAFs have been linked to immunosuppression, mainly by inhibiting T cell infiltration and activation [37][38][39][40][41], and tumor-infiltrating lymphocytes (TILs) were demonstrated to provide prognostic and treatment predictive information in breast cancer [3,42,43]. Potential prognostic and predictive effects of stromal PDGFRb expression in the primary tumor could be mediated by paracrine acting factors released by the microenvironment which act directly or indirectly on the tumor to promote progression and render tumor cells insensitive to RT.
Activation of PDGFRb, in particular on fibroblasts, has also been demonstrated to induce an upregulation of hepatocyte growth factor (HGF) and stanniocalcin-1 (STC1), with the latter having furthermore been linked to increased distant metastasis in several murine cancer models [18,[44][45][46]. In our study, the medium and high PDGFRb groups showed an increased propensity for any recurrence in univariable analysis, while no significant differences in rate of IBTR only were observed between the groups. In invasive breast cancer, a comprehensive IHC analysis approach recently identified four functional different fibroblast subsets [38], of which one subset was high in fibroblast activation protein (FAP) and PDGFRb expression and functionally linked to immunosuppression and pro-invasive effects [37,38]. Another subset, defined by CD29, alpha smooth muscle actin (ASMA) and also PDGFRb expression was assigned to pro-metastatic effects mostly through matrix remodeling [37]. A specific functional role of PDG-FRb expression was however not identified within these studies and it is unclear if the prognostic or potentially predictive effects are mediated directly by PDGFRb expressing cells through downstream-signaling or indirectly through an effect on other cells of the tumor microenvironment, such as TILs. In addition, PDGFRb could simply be a marker for a functional fibroblast or vascular mural cell subset with distinct effects on tumor progression. However, PDGFRb did not correlate positively with the overall stroma fraction. In our cohort, the overall stroma fraction was highest among Luminal A tumors and lowest among triple negative tumors. PDGFRb score showed the opposite distribution among subtypes and was instead correlated with unfavorable clinicopathological variables. We believe this can explain why PDGFRb and overall stroma fraction did not correlate. Previous studies have shown that a higher stroma fraction is associated with an unfavorable prognosis, particularly in triple negative tumors [47]. However, among ER positive tumors a higher stroma content has also been associated with favorable clinicopathological variables and with a better prognosis which conforms with our findings [48,49]. PDGFRb could simply be a marker for a functional fibroblast or vascular mural cell subset with distinct effects on tumor progression, and our study suggests that it may not correlate strongly with overall stroma. Recent singlecell sequencing data of stroma cells from a murine breast cancer model suggested the existence of specific vascular and matrix-remodeling CAF subsets [50]. It would therefore be of interest to further relate stromal PDGFRb-positivity to vessel density as well as abundance and composition of extracellular matrix. Additionally, studies applying multiplexed panels of markers for fibroblasts and pericytes as well as their activation status could provide a more specific definition of mesenchymal cell subsets as well as cell type stratification and thereby refine the findings of the presented study.
A strength of the presented study is the large patient number and the randomized design of the cohort allowing investigation of prognostic and predictive effects differentially. However, the study is to a certain extent limited through the utilization of a TMA format, which, although two cores per patient are included, may not sufficiently reflect heterogenous PDGFRb expression throughout the tumor tissue. Furthermore, PDGFRb scoring was performed manually by two independent raters and despite a moderate interrater agreement, unbiased digital approaches may be more sensitive. Initial digital approaches have been described [26,51,52] but are still under refinement given the fact that especially in early stage breast cancers, normal tissue regions very often are present and need to be excluded. Of note, in this retrospective study, the patients were categorized into PDG-FRb score groups based on tertiles as a predefined cut-off.
However, future studies using optimized cut-off strategies and independent validation cohorts would be highly warranted to further study the potential RT-predictive nature of PDGFRb expression.
In summary, our study suggests that higher stromal PDG-FRb expression is associated with an increased risk of any recurrence, which however can partly be explained by its association with estrogen receptor negativity and young age. Although a reduced RT benefit among patients with high PDGFRb was observed both in uni-as well as multivariable analysis, the interaction between PDGFRb and RT was not significant. Overall, the presented data motivates the experimental investigation of paracrine signaling initiated through stromal PDGFRb expression on tumor progression and resistance to RT.
Author contributions CS conception and study design, IHC staining, marker evaluation, major manuscript writing; AST data analysis, major manuscript writing; RJE marker evaluation; LAA supervision and layout of histological marker evaluation; PM, MF conception and study design, original tissue collection, clinical database assembly; EH conception and study design, clinical database assembly and maintenance, major analysis; AÖ conception and study design, study design, manuscript writing, financial support; PK conception and study design, original tissue collection, clinical database assembly, manuscript writing, financial support. All authors were involved in data interpretation. All authors read, commented and approved the final manuscript.
Funding Open access funding provided by University of Gothenburg. PK is supported by the Swedish state under the agreement between the Swedish government and the county councils, ALF-agreement Grant No. ALFGBG-716711, the Swedish Cancer Society Grant No. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.