BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. Methods: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC 50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. Results: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t 1/2 , ∼8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. Conclusions: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

Comparison of partial-breast irradiation and intraoperative radiation to whole-breast irradiation in early-stage breast cancer patients: a Kaplan-Meier-derived patient data meta-analysis.

PURPOSE: Partial breast irradiation (PBI) and intraoperative radiation (IORT) represent alternatives to whole breast irradiation (WBI) following breast conserving surgery. However, data is mixed regarding outcomes. We therefore performed a pooled analysis of Kaplan-Meier-derived patient data from randomized trials to evaluate the hypothesis that PBI and IORT have comparable long-term rates of ipsilateral breast tumor recurrence as WBI. METHODS: In February, 2023, PubMed, EMBASE and Cochrane Central were systematically searched for randomized phase 3 trials of early-stage breast cancer patients undergoing breast-conserving surgery with PBI or IORT as compared to WBI. Time-to-event outcomes of interest included ipsilateral breast tumor recurrence (IBTR), overall survival (OS) and distant disease-free survival (DDFS). Statistical analysis was performed with R Statistical Software. RESULTS: Eleven randomized trials comprising 15,460 patients were included; 7,675 (49.6%) patients were treated with standard or moderately hypofractionated WBI, 5,413 (35%) with PBI and 2,372 (15.3%) with IORT. Median follow-up was 9 years. PBI demonstrated comparable IBTR risk compared with WBI (HR 1.20; 95% CI 0.95-1.52; p = 0.12) with no differences in OS (HR 1.02; 95% CI 0.90-1.16; p = 0.70) or DDFS (HR 1.15; 95% CI 0.81-1.64; p = 0.43). In contrast, patients treated with IORT had a higher IBTR risk (HR 1.46; 95% CI 1.23-1.72; p < 0.01) compared with WBI with no difference in OS (HR 0.98; 95% CI 0.84-1.14; p = 0.81) or DDFS (HR 0.91; 95% CI 0.76-1.09; p = 0.31). CONCLUSION: For patients with early-stage breast cancer following breast-conserving surgery, PBI demonstrated no difference in IBTR as compared to WBI while IORT was inferior to WBI with respect to IBTR.

The Effect of Wire Versus Magnetic Seed Localization on Lumpectomy Cavity Size.

PURPOSE: Our purpose was to assess whether an association exists between surgical localization technique and lumpectomy cavity size on radiation therapy planning computed tomography (CT) scan. METHODS AND MATERIALS: A single-institution retrospective review was conducted of women undergoing breast conserving surgery with wire or magnetic seed guided lumpectomy followed by adjuvant radiation therapy from 2018 to 2021. Patients of a surgeon only performing 1 localization technique or undergoing bracketed localization were excluded. The primary outcome was lumpectomy cavity size on simulation CT. Confounding due to imbalance in patient and tumor factors was addressed with overlap weights derived from a propensity score analysis and used in a weighted multivariable analysis. Secondary outcomes included positive margins, total pathologic volume, boost delivery, and boost modality. RESULTS: Of 617 women who received lumpectomy during the study period, 387 were included in final analysis. Tumors of patients undergoing seed localization were more likely unifocal, assessable by ultrasound, and smaller. Seed use rates ranged from 27.7% to 70.7% per surgeon. There was no difference in positive margins (6.4 vs 5.4%, P = .79) or second surgeries (9.4 vs 8.1%, P = .79) between groups. Close margin rates were similar for ductal carcinoma in situ (P = .35) and invasive carcinoma (P = .97). In unadjusted bivariable analyses, wire localization was associated with larger total pathology volume (P = .004), but localization technique showed no association with CT cavity volume (P = .15). After adjusting for potentially confounding variables, multivariable analysis failed to show an association between localization technique and either CT cavity (P = .35) or total path volume (P = .08). There was no difference in indicated-boost delivery (P = .15) or electron boost (P = .14) by localization technique. CONCLUSIONS: There was no significant difference in CT cavity size by localization technique, suggesting choice between surgical techniques does not impede radiation therapy boost delivery.

Impact of sequencing of androgen receptor-signaling inhibition and radiotherapy in prostate cancer: importance of homologous recombination disruption.

PURPOSE: The synergy of combining androgen receptor-signaling inhibition (ARSI) to radiotherapy (RT) in prostate cancer has been largely attributed to non-homologous end joining (NHEJ) inhibition. However, this mechanism is unlikely to explain recently observed trial results that demonstrated the sequencing of ARSI and RT significantly impacts clinical outcomes, with adjuvant ARSI following RT yielding superior outcomes to neoadjuvant/concurrent therapy. We hypothesized this is driven by differential effects on AR-signaling and alternative DNA repair pathway engagement based on ARSI/RT sequencing. METHODS: We explored the effects of ARSI sequencing with RT (neoadjuvant vs concurrent vs adjuvant) in multiple prostate cancer cell lines using androgen-deprived media and validation with the anti-androgen enzalutamide. The effects of ARSI sequencing were measured with clonogenic assays, AR-target gene transcription and translation quantification, cell cycle analysis, DNA damage and repair assays, and xenograft animal validation studies. RESULTS: Adjuvant ARSI after RT was significantly more effective at killing colony forming cells and decreasing the transcription and translation of downstream AR-target genes across all prostate cancer models evaluated. These results were reproduced in xenograft studies. The differential effects of ARSI sequencing were not fully explained by NHEJ inhibition alone, but by the additional disruption of homologous recombination specifically with adjuvant sequencing of ARSI. CONCLUSION: We demonstrate that altered sequencing of ARSI and RT mediates differential anti-AR-signaling and anti-cancer effects, with the greatest benefit from adjuvant ARSI following RT. These results, combined with our prior clinical findings, support the superiority of an adjuvant-based sequencing approach when using ARSI with RT.

Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.

EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.

Radiotherapy Use and Incidence of Locoregional Recurrence in Patients With Favorable-Risk, Node-Positive Breast Cancer Enrolled in the SWOG S1007 Trial.

Importance: Little is known about regional nodal irradiation (RNI) practice patterns or rates of locoregional recurrence (LRR) with and without RNI in patients with limited nodal disease and favorable biology treated with modern surgical and systemic therapy, including approaches that de-escalate those latter treatments. Objective: To investigate how often patients with low-recurrence score breast cancer with 1 to 3 nodes involved receive RNI, incidence and predictors of LRR, and associations between locoregional therapy and disease-free survival. Design, Setting, and Participants: In this secondary analysis of the SWOG S1007 trial, patients with hormone receptor-positive, ERBB2-negative breast cancer, and a Oncotype DX 21-gene Breast Recurrence Score assay result of no more than 25, were randomized to endocrine therapy alone vs chemotherapy then endocrine therapy. Prospectively collected radiotherapy information was collected from 4871 patients treated in diverse settings. Data were analyzed June 2022 to April 2023. Exposure: Receipt of RNI (targeting at least the supraclavicular region). Main Outcome(s) and Measure(s): Cumulative incidence of LRR was calculated by locoregional treatment received. Analyses were assessed for associations between invasive disease-free survival (IDFS) and locoregional therapy, adjusted for menopausal status, treatment group, recurrence score, tumor size, nodes involved, and axillary surgery. Radiotherapy information was recorded in the first year after randomization, so survival analyses were landmarked as starting at 1 year among those still at risk. Results: Of 4871 female patients (median [range] age, 57 [18-87] years) with radiotherapy forms, 3947 (81.0%) reported radiotherapy receipt. Of 3852 patients who received radiotherapy and had complete information on targets, 2274 (59.0%) received RNI. With a median follow-up of 6.1 years, the cumulative incidence of LRR by 5 years was 0.85% among patients who received breast-conserving surgery and radiotherapy with RNI; 0.55% after breast-conserving surgery with radiotherapy without RNI; 0.11% after mastectomy with postmastectomy radiotherapy; and 1.7% after mastectomy without radiotherapy. Similarly low LRR was observed within the group assigned to endocrine therapy without chemotherapy. The rate of IDFS did not differ by RNI receipt (premenopausal: hazard ratio [HR], 1.03; 95% CI, 0.74-1.43; P = .87; postmenopausal: HR, 0.85; 95% CI, 0.68-1.07; P = .16). Conclusions and Relevance: In this secondary analysis of a clinical trial, RNI use was divided in the setting of biologically favorable N1 disease, and rates of LRR were low even in patients who did not receive RNI. Disease-free survival was not associated with RNI receipt; omission of chemotherapy among patients similar to those enrolled in the S1007 trial is not an independent indication for use of RNI.

Integrating Tumor-Intrinsic and Immunologic Factors to Identify Immunogenic Breast Cancers from a Low-Risk Cohort: Results from the Randomized SweBCG91RT Trial.

PURPOSE: The local immune infiltrate's influence on tumor progression may be closely linked to tumor-intrinsic factors. The study aimed to investigate whether integrating immunologic and tumor-intrinsic factors can identify patients from a low-risk cohort who may be candidates for radiotherapy (RT) de-escalation. EXPERIMENTAL DESIGN: The SweBCG91RT trial included 1,178 patients with stage I to IIA breast cancer, randomized to breast-conserving surgery with or without adjuvant RT, and followed for a median of 15.2 years. We trained two models designed to capture immunologic activity and immunomodulatory tumor-intrinsic qualities, respectively. We then analyzed if combining these two variables could further stratify tumors, allowing for identifying a subgroup where RT de-escalation is feasible, despite clinical indicators of a high risk of ipsilateral breast tumor recurrence (IBTR). RESULTS: The prognostic effect of the immunologic model could be predicted by the tumor-intrinsic model (Pinteraction = 0.01). By integrating measurements of the immunologic- and tumor-intrinsic models, patients who benefited from an active immune infiltrate could be identified. These patients benefited from standard RT (HR, 0.28; 95% CI, 0.09-0.85; P = 0.025) and had a 5.4% 10-year incidence of IBTR after irradiation despite high-risk genomic indicators and a low frequency of systemic therapy. In contrast, high-risk tumors without an immune infiltrate had a high 10-year incidence of IBTR despite RT treatment (19.5%; 95% CI, 12.2-30.3). CONCLUSIONS: Integrating tumor-intrinsic and immunologic factors may identify immunogenic tumors in early-stage breast cancer populations dominated by ER-positive tumors. Patients who benefit from an activated immune infiltrate may be candidates for RT de-escalation.

Subclonal evolution and expansion of spatially distinct THY1-positive cells is associated with recurrence in glioblastoma.

PURPOSE: Glioblastoma(GBM) is a lethal disease characterized by inevitable recurrence. Here we investigate the molecular pathways mediating resistance, with the goal of identifying novel therapeutic opportunities. EXPERIMENTAL DESIGN: We developed a longitudinal in vivo recurrence model utilizing patient-derived explants to produce paired specimens(pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR). These specimens were evaluated for treatment response and to identify gene expression pathways driving treatment resistance. Findings were clinically validated using spatial transcriptomics of human GBMs. RESULTS: These studies reveal in replicate cohorts, a gene expression profile characterized by upregulation of mesenchymal and stem-like genes at recurrence. Analyses of clinical databases revealed significant association of this transcriptional profile with worse overall survival and upregulation at recurrence. Notably, gene expression analyses identified upregulation of TGFß signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Furthermore, THY1-positive cells represented <10% of cells in treatment-naïve tumors, compared to 75-96% in recurrent tumors. We then isolated THY1-positive cells from treatment-naïve patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naïve human GBM, we conducted spatial transcriptomic analyses. This revealed rare THY1+ regions characterized by mesenchymal/stem-like gene expression, analogous to our recurrent mouse model, which co-localized with macrophages within the perivascular niche. We then inhibited TGFBRI activity in vivo which decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors. CONCLUSIONS: These findings reveal that GBM recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive, mesenchymal cells within the perivascular niche.

Evaluation of the Sensitivity to Endocrine Therapy Index and 21-Gene Breast Recurrence Score in the SWOG S8814 Trial.

PURPOSE: Chemotherapy has not demonstrated benefit over adjuvant endocrine therapy alone for postmenopausal patients with node-positive breast cancer with a 21-gene breast recurrence score (RS) of 25 or below (RS ≤ 25). We tested whether combined results from RS and the sensitivity to endocrine therapy (SET2,3) index of endocrine-related transcription (SETER/PR) adjusted for baseline prognostic index (BPI) improve prognostic assessment, and whether SET2,3 predicted benefit from anthracycline-based chemotherapy. METHODS: A blinded retrospective clinical validation of SET2,3 in two randomized treatment arms from the SWOG S8814 trial comparing adjuvant anthracycline-based chemotherapy followed by tamoxifen endocrine therapy for 5 years, versus tamoxifen alone. SET2,3 assay was calibrated and measured using whole-transcriptome RNA sequence of tumor samples already tested for RS. The primary end point was disease-free survival (DFS). RESULTS: There were 106 events in 283 patients over a median follow-up of 8.99 years. Proportional hazards assumptions were met during the first 5 years only. SET2,3 index and RS were not correlated (r = -0.04) and were independently prognostic (SET2,3: hazard ratio [HR], 0.48 per unit; 95% CI, 0.34 to 0.68; P < .001; RS: HR, 1.28 per 10 units; 95% CI, 1.14 to 1.44; P < .001). SET2,3 index did not predict chemotherapy benefit (interaction P = .77). SET2,3 was high in 93/175 (53%) patients with RS ≤ 25 (concordant low-risk), with 5-year DFS 97%. SET2,3 was low in 55/108 (51%) patients with RS > 25 (concordant high-risk), with 5-year DFS 53%. Both components of SET2,3 index were prognostic after adjustment for RS: SETER/PR (HR, 0.65; 95% CI, 0.46 to 0.92) and BPI (HR, 0.45; 95% CI, 0.31 to 0.64). CONCLUSION: SET2,3 index was not correlated with RS, demonstrated additive prognostic performance, and was not chemopredictive in this subset of patients from S8814. The SETER/PR and BPI components of SET2,3 each added prognostic information to RS.

Development and Validation of a Genomic Profile for the Omission of Local Adjuvant Radiation in Breast Cancer.

PURPOSE: Adjuvant radiotherapy (RT) is used for women with early-stage invasive breast cancer treated with breast-conserving surgery. However, some women with low risk of recurrence may safely be spared RT. This study aimed to identify these women using a molecular-based approach. METHODS: We analyzed two randomized trials of women with node-negative invasive breast cancer to ± RT following breast-conserving surgery: SweBCG91-RT (stage I-II, no adjuvant systemic therapy) and Princess Margaret (age 50 years or older, T1-T2, adjuvant tamoxifen). Transcriptome-wide profiling was performed (Affymetrix Human Exon 1.0 ST microarray). Patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative tumors and with gene expression data were included. The SweBCG91-RT cohort was divided into training (N = 243) and validation (N = 354) cohorts. A 16-gene signature named Profile for the Omission of Local Adjuvant Radiation (POLAR) was trained to predict locoregional recurrence (LRR) using elastic net regression. POLAR was then validated in the SweBCG91-RT validation cohort and the Princess Margaret cohort (N = 132). RESULTS: Patients categorized as POLAR low-risk without RT had a 10-year LRR of 6% (95% CI, 2 to 16) and 7% (0 to 27) in SweBCG91-RT and Princess Margaret cohorts, respectively. There was no significant benefit from RT in POLAR low-risk patients (hazard ratio [HR], 1.1 [0.39 to 3.4], P = .81, and HR, 1.5 [0.14 to 16], P = .74, respectively). Patients categorized as POLAR high-risk had a significant decreased risk of LRR with RT (HR, 0.43 [0.24 to 0.78], P = .0055, and HR, 0.25 [0.07 to 0.92], P = .038, respectively). An exploratory analysis testing for interaction between RT and POLAR in the combined validation cohort was performed (P = .066). CONCLUSION: The novel POLAR genomic signature on the basis of LRR biology may identify patients with a low risk of LRR despite not receiving RT, and thus may be candidates for RT omission.

Bcl-xL inhibition radiosensitizes PIK3CA/PTEN wild-type triple negative breast cancers with low Mcl-1 expression.

Patients with radioresistant breast cancers, including a large percentage of women with triple negative breast cancer (TNBC), demonstrate limited response to radiation (RT) and increased locoregional recurrence; thus, strategies to increase the efficacy of RT in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant PIK3CA/PTEN TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of PIK3CA/PTEN status (rER: 0.95 - 1.07). In wild-type PIK3CA/PTEN TNBC, pan Bcl-2 family inhibition or Bcl-xL specific inhibition with RT led to increased levels of apoptosis (p < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated PTEN knockout in wild-type PIK3CA/PTEN MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor-mediated radiosensitization (rER: 0.94 - 1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02 - 1.04) but transient MCL1 knockdown in CAL-51 cells promoted Bcl-xL-inhibitor mediated radiosensitization (rER 2.35 ± 0.05). In vivo, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence.

Updates in combined approaches of radiotherapy and immune checkpoint inhibitors for the treatment of breast cancer.

Breast cancer is the most prevalent non-skin cancer diagnosed in females and developing novel therapeutic strategies to improve patient outcomes is crucial. The immune system plays an integral role in the body's response to breast cancer and modulating this immune response through immunotherapy is a promising therapeutic option. Although immune checkpoint inhibitors were recently approved for the treatment of breast cancer patients, not all patients respond to immune checkpoint inhibitors as a monotherapy, highlighting the need to better understand the biology underlying patient response. Additionally, as radiotherapy is a critical component of breast cancer treatment, understanding the interplay of radiation and immune checkpoint inhibitors will be vital as recent studies suggest that combined therapies may induce synergistic effects in preclinical models of breast cancer. This review will discuss the mechanisms supporting combined approaches with radiotherapy and immune checkpoint inhibitors for the treatment of breast cancer. Moreover, this review will analyze the current clinical trials examining combined approaches of radiotherapy, immunotherapy, chemotherapy, and targeted therapy. Finally, this review will evaluate data regarding treatment tolerance and potential biomarkers for these emerging therapies aimed at improving breast cancer outcomes.

Tailoring the Omission of Radiotherapy for Early-Stage Breast Cancer Based on Tumor Biology.

Advances in our understanding of tumor biology now permit greater personalization of radiotherapy for early-stage breast cancer. De-escalation of radiotherapy is an important opportunity that exists due to improvements in breast imaging, surgery, pathology, and systemic therapy. These modern tools, in addition to molecular subtype and prediction assays, have allowed us to select certain patients with breast cancer based on favorable biology who are at sufficiently low-risk of local recurrence that they may reasonably consider the omission of radiotherapy to reduce toxicity and burden. For decades, clinical trials have been conducted to try to identify the population of patients in whom the risk of recurrence in the absence of radiotherapy is sufficiently small that omission of adjuvant radiotherapy might acceptably be considered after breast conserving surgery. Trials to date have largely included patients with estrogen receptor-positive breast cancer selected based upon clinicopathologic characteristics alone and have yet to identify a patient population for whom radiotherapy is not effective. They have, however, identified women with lower absolute risks of locoregional recurrence with radiotherapy omission, and current guidelines do recognize this patient population as candidates for radiotherapy omission after breast conserving surgery. Studies that assess not just clinicopathologic features but predictive molecular biomarkers of the underlying biology are currently ongoing. This manuscript will briefly review historical trials evaluating the role of radiotherapy after breast conserving surgery as well as discuss the exciting ongoing trials in hormone receptor-positive disease leveraging our growing appreciation of tumor biology and molecular prediction to identify a population of patients who may reasonably consider omitting radiotherapy after breast conserving surgery.

Androgen and oestrogen receptor co-expression determines the efficacy of hormone receptor-mediated radiosensitisation in breast cancer.

PURPOSE: Radiation therapy (RT) and hormone receptor (HR) inhibition are used for the treatment of HR-positive breast cancers; however, little is known about the interaction of the androgen receptor (AR) and estrogen receptor (ER) in response to RT in AR-positive, ER-positive (AR+/ER+) breast cancers. Here we assessed radiosensitisation of AR+/ER+ cell lines using pharmacologic or genetic inhibition/degradation of AR and/or ER. METHODS: Radiosensitisation was assessed with AR antagonists (enzalutamide, apalutamide, darolutamide, seviteronel, ARD-61), ER antagonists (tamoxifen, fulvestrant) or using knockout of AR. RESULTS: Treatment with AR antagonists or ER antagonists in combination with RT did not result in radiosensitisation changes (radiation enhancement ratios [rER]: 0.76-1.21). Fulvestrant treatment provided significant radiosensitisation of CAMA-1 and BT-474 cells (rER: 1.06-2.0) but not ZR-75-1 cells (rER: 0.9-1.11). Combining tamoxifen with enzalutamide did not alter radiosensitivity using a 1 h or 1-week pretreatment (rER: 0.95-1.14). Radiosensitivity was unchanged in AR knockout compared to Cas9 cells (rER: 1.07 ± 0.11), and no additional radiosensitisation was achieved with tamoxifen or fulvestrant compared to Cas9 cells (rER: 0.84-1.19). CONCLUSION: While radiosensitising in AR + TNBC, AR inhibition does not modulate radiation sensitivity in AR+/ER+ breast cancer. The efficacy of ER antagonists in combination with RT may also be dependent on AR expression.

Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models.

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51-0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50-1.60, fulvestrant rER: 1.76-2.81, AZD9496 rER: 1.33-1.48, estrogen depletion rER: 1.47-1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

RB expression confers sensitivity to CDK4/6 inhibitor-mediated radiosensitization across breast cancer subtypes.

Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor-positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49-2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84-1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88-1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.