Mutter, Robert W.

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5366.

Abstract INTRODUCTION Accelerated atherosclerosis has been described in breast cancer patients receiving cardiac exposure from adjuvant radiotherapy (RT). As a result of multidisciplinary care, breast cancer cure rates have improved in recent decades. Therefore, evaluating radiation-induced heart disease (RIHD), especially coronary artery disease (CAD), is essential. The 2022 cardio-oncology European Society of Cardiology (ESC) guidelines recommend screening patients 5 to 10 years after RT for CAD according to cardiovascular risk factors. This screening is generally recommended with exercise stress echocardiography since it can assess the physiological functional performance. Until now, it is unknown if dobutamine stress echocardiography (DSE) is an equal screening tool and if it can predict major adverse cardiovascular events (MACE) (myocardial infarction, stroke, cardiac death, revascularization). PURPOSE Evaluate if DSE is an adequate tool to stratify and predict MACE in post-radiotherapy patients. Methods A retrospective, descriptive cohort study was conducted. Patients who had chest RT for breast cancer and underwent DSE from 2000 to 2022 were included. A bivariate analysis, including Kruskal Wallis and Pearson Chi-Squared test, was done to identify variables associated with a positive DSE. A multivariable Cox Model for MACE and a time-to-event curve for MACE were also performed. Results We identified 163 patients that underwent DSE after a median time of 5.8 years from the end of RT. The median age was 74 years, 98.2% were female, 50% had left and 45% right breast cancer, 81% had hypertension, 41% diabetes mellitus, 71% dyslipidemia, 34% were smokers, and 19% had a prior myocardial infarction. The average dose of photon RT to the chest was 55Gy in the cohort, and 40% received chemotherapy before RT. Patients were divided into two groups: positive and negative DSE. A positive DSE was seen in 19.6%. The median (Q1, Q3) regional wall motion abnormalities (RWMA) of patients with a positive DSE was 1 (1, 1.015) at baseline and 1.280 (1.190, 1.520) at peak stress, and for a negative DSE, was 1 (1, 1) at baseline and peak stress. 88.2% of negative DSE and 87.5% of positive DSE achieved the target heart rate of 85%. HTN, dyslipidemia, and prior MI were significantly associated with a positive DSE (p <0.023). The cumulative incidence of MACE was higher in the group of positive DSE (p<0.001), even after adjusting for hypertension, diabetes mellitus, dyslipidemia, smoking, and prior myocardial infarction, with a hazard ratio of 2.500 (1.51 – 4.13) and a p< 0.001. Similar results were observed regardless of RT laterality. Conclusions In breast cancer survivors that received adjuvant radiotherapy as part of multidisciplinary care, a positive DSE was an independent predictor of MACE compared to a negative result. These results support the usefulness of dobutamine stress echocardiography as a screening tool in breast cancer survivors.Kaplan-Meier Curve for MACE

Abstract Background Exercise stress echocardiography (ESE) is a non-invasive, radiation-free, and widely available technique that has demonstrated a reliable accuracy for the detection of coronary artery disease (CAD). Chest radiotherapy (RT) is associated with accelerated age-related atherosclerosis and fibrosis in a dose-dependent manner. ESE plays a role as a screening tool of RT induced heart disease, however, the prognostic value of changes in regional wall motion abnormalities in this population during ESE has not been evaluated. Methods This is a retrospective cohort study of breast cancer survivors treated with adjuvant RT that underwent ESE from 2000-2020 after treatment completion. Outcomes evaluated were: major adverse cardiovascular events (MACE: cardiovascular death, myocardial infarction, revascularization and stroke); and all-cause mortality. A univariate analysis was performed to characterize the population. Continuous variables were presented as means ± standard deviation (SD) or median with quartile 1 and 3 (Q1, Q3), and categorical variables as numbers and percentages. A Cox regression model adjusted by age and cardiovascular risk factors was used to evaluate variables associated with outcomes, and a time to first event approach was used. A p-value of <0.05 was considered statistically significant. Results From 2000-2020, 507 patients, with a mean age 66 ± 10 years, underwent ESE at a median of 5.8 years after RT completion. The most prevalent comorbidities were dyslipidemia (59%), HTN (55%) and obesity (37%). 55% underwent RT for left-sided breast cancer and 45% for right-sided, and median RT dose was 6000 cGy. Baseline characteristics are presented in Table 1. Most patients underwent Bruce Protocol (92%). Patients exercised a mean time of 6.6 minutes achieving 7.3 METS and 109% of functional aerobic capacity (Table 2). At a median follow up of 7.8 years, 61 MACE events occurred. Predictors of MACE were double product, baseline and stress EF, baseline and peak regional wall motion abnormalities (RWMA), development of new ischemic RWMA, inability to reach 85% of maximum HR, and an abnormal EF response with stress after adjusting for age, HTN, DM, smoking history, dyslipidemia, obesity, prior MI and cancer staging (Fig 1). Predictors of all-cause mortality were METS, LV mass index, baseline and peak RWMA, development of new ischemic RWMA and an abnormal EF change with stress even after adjusting for variables described previously (Fig 2). Conclusions The development of new or worsening RWMA of just 2 segments with stress, was a significant predictor of MACE and all-cause mortality in a large cohort of breast cancer survivors undergoing multidisciplinary management including adjuvant RT. Special emphasis on management of cardiovascular comorbidities and closer surveillance are recommended for these patients.

AbstractBackground and ObjectivesCurrent NCCN guidelines discourage repeat sentinel lymph node (SLN) surgery in patients with local recurrence (LR) of breast cancer following prior mastectomy. This study addresses the feasibility and therapeutic impact of this approach.MethodsWe identified 73 patients managed with repeat SLN surgery for post−mastectomy isolated LR. Lymphatic mapping was performed using radioisotope with or without lymphoscintigraphy and/or blue dye. Successful SLN surgery was defined as retrieval of ≥1 SLN.ResultsSLN surgery was successful in 65/73 (89%), identifying a median of 2 (range 1–4) SLNs, with 10/65 (15%) SLN‐positive. Among these, 5/10 (50%) proceeded to ALND. In unsuccessful cases, 1/8 (13%) proceeded to ALND. Seven of 10 SLN‐positive patients and 50/55 SLN‐negative patients received adjuvant radiotherapy. Chemotherapy was administered in 31 (42%) and endocrine therapy in 50 of 57 HR+ patients (88%). After 28 months median follow‐up, eight patients relapsed with the first site local in two, distant in five, and synchronous local/distant in one. No nodal recurrences were observed.ConclusionsSLN surgery for patients with LR post−mastectomy is feasible and informative. This approach appears oncologically sound, decreases axillary dissection rates and may be used to tailor adjuvant radiation target volumes and systemic therapies.

1. Background: We sought to determine acute and subacute changes in cardiac function after proton beam (PBT) and photon beam (PhT) radiotherapy (RT) using conventional and two-dimensional speckle tracking echocardiography (2D-STE) in patients with malignant breast and thoracic tumors. 2. Methods: Between March 2016 and March 2017, 70 patients with breast or thoracic cancer were prospectively enrolled and underwent transthoracic echocardiography with comprehensive strain analysis at pretreatment, mid-treatment, end of treatment, and 3 months after RT. 3. Results: PBT was used to treat 44 patients; PhT 26 patients. Mean ± SD age was 55 ± 12 years; most patients (93%) were women. The median (interquartile range) of the mean heart dose was lower in the PBT than the PhT group (47 [27–79] vs. 217 [120–596] cGy, respectively; p < 0.001). Ejection fraction did not change in either group. Only the PhT group had reduced systolic tissue Doppler velocities at 3 months. 2D-STE showed changes in endocardial and epicardial longitudinal, radial, and circumferential early diastolic strain rate (SRe) in patients undergoing PhT (global longitudinal SRe, pretreatment vs. end of treatment (p = 0.04); global circumferential SRe, pretreatment vs. at 3-month follow-up (p = 0.003); global radial SRe, pretreatment vs. at 3-month follow-up (p = 0.02) for endocardial values). Epicardial strain values decreased significantly only in patients treated with PhT. Patients in the PhT group had a significant decrease in epicardial global longitudinal systolic strain rate (GLSRs) (epicardial GLSRs, at baseline vs. at end of treatment [p = 0.009]) and in GCSRe and GRSRe (epicardial GCSRe, at baseline vs. at 3-month follow-up (p = 0.02); epicardial GRSRe, at baseline vs. at 3-month follow-up (p = 0.03)) during treatment and follow-up. No changes on 2D-STE were detected in the PBT group. 4. Conclusions: Patients who underwent PhT but not PBT had reduced tissue Doppler velocities and SRe values during follow-up, suggesting early myocardial relaxation abnormalities. PBT shows promise as a cardiac-sparing RT technology.

Abstract PD-L1/PD-1 receptor pathway represents an important way of cancer cells to escape immune surveillance, yet only small portions of patients can benefit from PD-1/PD-L1 inhibitors. This is at least partially due to incomplete understanding of PD-1/PD-L1 biology in cancer. Although the interaction of extracellular domains between PD-L1 and PD-1 on cancer cells and immune cells is well studied, the function of PD-L1 inside cells, however, has not been well established. In our previous data, we found that intracellular PD-L1 acts as an RNA binding protein and interacts with NBS1 and BRCA1 mRNAs. Furthermore, intracellular PD-L1 protects targeted RNAs from degradation by RNA exosome, therefore increasing cellular resistance to DNA damage. Since Clinical approved PD-1/PD-L1 inhibitors have little effect on intracellular PD-L1, we sought to find new drugs to target intracellular PD-L1. Here we developed a new PD-L1 antibody, H1A, which has a different effect on PD-L1. Instead of blocking of PD-1/PD-L1 interaction, we found that this antibody disrupts the interaction of PD-L1 and CMTM6, which leads to degradation of PD-L1 by the lysosome. Therefore, H1A can unleash host antitumor activity and enhance the effects of DNA damaging therapy in cancer cells at the same time. Indeed, applying H1A in vivo significantly sensitized tumors to radiation and chemotherapy in a PDX models. Moreover, in a humanized immune competent TNBC PDX model, H1A monotherapy exhibited better therapeutic efficacy compared with clinically available PD-L1 inhibitor, suggesting that H1A activates anti-tumor immunity through PD-L1 degradation. Therefore, H1A may be a potential new drug to target both intracellular and extracellular PD-L1 to improve cancer therapy.

Introduction: Exercise Stress Echocardiography (ESE) is recommended as a screening tool for the evaluation of Coronary Artery Disease (CAD) in patients with suspected Radiation-Induced Heart Disease (RIHD). Up to now, studies have only evaluated its association with the extent of CAD. Hypothesis Cancer survivors treated with chest Radiotherapy (RT) that undergo an ESE and have a +ESE develop more MACE than those who have -ESE. Methods: A retrospective, descriptive, cohort study was conducted. Patients who had chest RT and underwent ESE with Treadmill Bruce Stress Protocol, from 2000 to 2012, at Mayo Clinic Rochester and Mayo Clinic Health System were included. A univariate analysis was performed to characterize the population. An analysis including Kruskal Wallis and Pearson Chi-Squared tests was completed to identify variables associated with + SE (Table 1). Multivariable Cox Model for MACE was conducted and is shown in Table 2. A time-to-event curve using Kaplan-Meier estimates is shown in Figure 1. Results: We identified 113 patients, with a mean age of 67 years and a median follow-up of 15.1 years. Of those, 99% were female, 98% were breast cancer survivors, 59% had HTN, 14% DM, 11% AFib, 2% COPD, and 12% had a history of MI. All the patients received >3000cGy of Photon RT, and 57% were treated with systemic cancer therapies. A +ESE was seen in 20.3% of the patients with no significant difference in METS achieved compared with patients who had a -ESE. COPD, RT dose, and systemic therapies, specifically doxorubicin, were associated with a +ESE. The cumulative incidence of MACE was higher in the group of +ESE (p=0.029). After adjustment for HTN, DM, smoking history, hyperlipidemia, and prior MI, the HR for MACE associated with a +ESE was 1.97 (1.09-3.59). Conclusion: MACE was more frequent in patients with a +ESE who received chest RT and doxorubicin versus -ESE. These results support the usefulness of ESE in cancer survivors after RT as a cardiovascular screening tool.

Introduction: patients exposed to chest, breast or mediastinal RT, may present with accelerated atherosclerosis and CAD due to diffuse micro and macrovascular fibrosis. However, CAD risk has not been well established in a community-based study. Aims: Describe CAD risk in patients treated with chemotherapy with and without RT and evaluate the cumulative incidence of CAD (coronary revascularization, MI and abnormal stress test and coronary angiogram). Methods: From 1985-2010, we identified 744 patients with a new diagnosis of breast cancer or lymphoma, using the Rochester Epidemiology Project (REP) who received chemotherapy with or without RT. CAD risk was assessed by a proportional hazards model for time to event. RT was treated as a time dependent co-variable, with index date being chemotherapy start date, significance was considered as a p<.05. Characteristics were evaluated by a multivariable Cox model to consider RT and time censoring. Results: 744 patients treated with chemotherapy were identified, of these 488 (65.6%) had breast cancer, 256 (34.4%) lymphoma, and 367 (49%) also received RT. The mean total RT dose was 5.5 ± 1.2 (Gy) for those that had CAD and 5.4 (Gy) ± 1.1 for non-CAD patients, p=0.4. The cumulative incidence of CAD events (accounting for death as a competing risk) at 5 and 10 years of follow-up was 1.9% (CI 1.14-3.2) and 3.06% (CI 2.01-4.7), respectively p<.05 figure1. Baseline differences between those receiving RT or not, were not significant for DM and HTN; however, males and ages 70-79 were less likely to receive RT, table1. In a multivariable model, there was no difference in the CAD outcomes between those with and without RT, HR= 1.21 [0.48-3.06] p=.69, after adjusting for age, gender, DM, and HTN and those <49 y/o had a lower risk of CAD. Table2 Conclusion: In this REP retrospective cohort study, the risk of developing a CAD event was similar between chemotherapy plus RT and chemotherapy only regimens, even after considering gender, age, HTN and DM.

Abstract Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase known to regulate immune cell function, cell adhesion, and vascular development. Here, we report that Syk can be expressed in high grade serous ovarian cancer and triple negative breast cancers and promotes DNA double strand break resection, homologous recombination (HR) and therapeutic resistance. We found that Syk is activated by ATM following DNA damage and is recruited to DNA double strand breaks by NBS1. Once at the break site, Syk phosphorylates CtIP, a key mediator of resection and HR, at Thr-847 to promote repair activity, specifically in Syk expressing cancer cells. Syk inhibition or genetic deletion abolished CtIP Thr-847 phosphorylation and overcame the resistant phenotype. Collectively, our findings suggest that Syk drives therapeutic resistance by promoting DNA resection and HR through a novel ATM-Syk-CtIP pathway, and that Syk is a new tumor-specific target to sensitize Syk-expressing tumors to PARPi and other DNA targeted therapy.

Abstract Purpose: Triple-negative invasive lobular carcinoma (TN-ILC) of breast cancer is a rare disease and the clinical outcomes and prognostic factors are not well-defined. Methods: Women with stage I-III TN-ILC or triple-negative invasive ductal carcinoma (TN-IDC) of the breast undergoing mastectomy or breast-conserving surgery between 2010 and 2018 in the National Cancer Database were included. Kaplan-Meier curves and multivariate Cox proportional hazard regression were used to compare overall survival (OS) and evaluate prognostic factors. Multivariate logistic regression was performed to analyze the factors associated with pathological response to neoadjuvant chemotherapy. Results: The median age at diagnosis for women with TN-ILC was 67 years compared to 58 years in TN-IDC (p<0.001). There was no significant difference in the OS between TN-ILC and TN-IDC in multivariate analysis (HR 0.96, p=0.44). Black race and higher TNM stage were associated with worse OS, whereas receipt of chemotherapy or radiation was associated with better OS in TN-ILC. Among women with TN-ILC receiving neoadjuvant chemotherapy, the 5-year OS was 77.3% in women with a complete pathological response (pCR) compared to 39.8% in women without any response. The odds of achieving pCR following neoadjuvant chemotherapy were significantly lower in women with TN-ILC compared to TN-IDC (OR 0.53, p<0.001). Conclusion: Women with TN-ILC are older at diagnosis but have similar OS compared to TN-IDC after adjusting for tumor and demographic characteristics. Administration of chemotherapy was associated with improved OS in TN-ILC, but women with TN-ILC were less likely to achieve complete response to neoadjuvant therapy compared to TN-IDC.