Patients with Fanconi anemia (FA) are particularly susceptible to developing squamous cell carcinoma of the head and neck due to impaired DNA repair pathways. However, their hypersensitivity to DNA damaging agents can limit effective treatment with standard radiotherapy due to severe side effects and complications. In pre-clinical models, ultra-rapid FLASH radiotherapy (FLASH) reduces radiation-induced toxicity in normal tissues while maintaining similar tumor control compared to conventional dose rate radiotherapy (CONV). Here, we investigated the safety of FLASH for treatment of the head and neck in a mouse model of FA. 129/Sv wild-type (WT) and Fanca-deficient (Fanca -/-) mice received single-dose oral cavity irradiation with electron beam FLASH or CONV to evaluate radiation-induced toxicity in non-tumor-bearing mice. Fanca WT and Fanca -/- mice were irradiated with 25 and 18 Gy, respectively, of FLASH (190 Gy/sec) or CONV (0.2 Gy/sec), with tongues harvested at 12 hours (hpi) and 10 days (dpi) post-irradiation. At 10 dpi, FLASH-irradiated tongues in both genetic backgrounds demonstrated reduced ulceration at the dorsal tongue surface compared to CONV-irradiated counterparts. Histopathological analysis of the tongue revealed lower mucositis severity scores with decreased epithelial thinning and ulceration in FLASH-irradiated tongues compared to CONV-irradiated ones. Analysis of γ-H2AX foci formation at 12 hpi demonstrated fewer foci in WT mice treated with FLASH compared to CONV, with a similar trend observed in Fanca -/- mice. These findings suggest a potential normal tissue-sparing effect with FLASH and hold important clinical implications for the treatment of patients with Fanconi anemia and head and neck cancers.

Abstract Background: Radiotherapy is central to breast cancer treatment but is limited by acute and cumulative skin toxicity, especially in large-field treatments and re-irradiation. Ultra-high-dose-rate FLASH radiotherapy (FLASH-RT, ≥ 40 Gy/s) may widen the therapeutic window by reducing normal-tissue injury without compromising tumor control. However, breast-directed and patient-derived models remain underexplored, including the effects of FLASH under repeated-irradiation conditions. Methods: We integrated an orthotopic triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model and hemithoracic normal-tissue models to compare FLASH-RT (180 Gy/s) with CONV-RT (0.03 Gy/s). TNBC-bearing NRG mice received single-fraction of 30Gy electron irradiation via a custom stereotactic jig enabling mammary-targeted or hemithoracic fields. A separate cohort of non-tumor-bearing NRG mice underwent left-chest re-irradiation to assess cumulative tolerance. Endpoints included tumor regression, recurrence, survival, and graded skin toxicity; ongoing analyses incorporate histopathology and single-cell/spatial transcriptomics to elucidate FLASH-mediated tissue responses and mechanisms of normal-tissue sparing. Results: FLASH-RT achieved equivalent tumor control to CONV-RT in TNBC-PDX models, with both modalities inducing complete regression by day 16 and maintaining clearance for two weeks before recurrence at day 32 post-RT. In contrast, normal-tissue responses diverged markedly: FLASH-RT significantly reduced acute skin toxicity (median score 0 vs. 5; p<0.0001), eliminated ulceration, and extended survival (120 vs. 90 days post-implantation) in tumor bearing mice. In non-tumor-bearing NRG mice, FLASH-RT also improved tolerance to cumulative thoracic irradiation; mice receiving a second 25Gy left-chest FLASH irradiation dose showed no clinical decline, whereas CONV-RT animals developed progressive toxicity requiring euthanasia within three months of re-irradiation. Multi-omics analyses are underway to define mechanisms of early tissue sparing and improved re-irradiation response. Conclusions: FLASH-RT maintains tumor-control efficacy equivalent to CONV-RT while significantly reducing skin toxicity in TNBC-PDX models and improving normal-tissue tolerance to re-irradiation in non-tumor-bearing NRG mice. These findings support FLASH-RT as a clinically promising strategy that may expand safe re-treatment options and broaden curative radiotherapy opportunities in breast cancer. Mechanistic studies are ongoing to elucidate the biological basis of early tissue sparing and guide translation into breast-conserving and post-mastectomy treatment settings. Citation Format: Adel Zaid I Mutahar, Banita Verma, Stavros Melemenidis, Suparna Dutt, Kerriann M. Casey, Zhen Qi, Angera Hsiao-Chi Kuo, Kathleen C. Horst, Edward Elliot Graves, Michael F. Clarke, Billy W. Loo, Frederick M. Dirbas. FLASH radiotherapy maintains tumor control and enables safe re-irradiation while preserving normal tissue in breast cancer PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5266.

Abstract Background: Radiotherapy (RT) is central to breast cancer (BC) management but limited by normal tissue toxicity. Conventional RT (CONV; ≤0.03 Gy/s) controls tumors but often causes skin inflammation, compromises treatment intensity and quality of life. Ultra-high dose-rate FLASH RT (>40 Gy/s) achieves comparable tumor control with markedly reduced tissue injury (“FLASH effect”), yet its mechanism of tumor control and sparing of normal tissue remains unclear. Ionizing radiation activates the cGAS-STING pathway through DNA damage, triggering proinflammatory cytokine production and tissue injury, yet has also been implicated with improvement in the antitumor immune response. Emerging data suggest that FLASH may attenuate or abrogate cGAS-STING signaling leading to reduced inflammation and tissue injury. This study investigated the role of cGAS signaling in mediating the differential effects of FLASH and CONV RT on normal tissue toxicity and tumor control using wild-type (WT) C57BL/6 and cGAS double knockout (cGAS -/- KO) mice. Methods: PYMT117 BC cells were orthotopically implanted into the third mammary fat pad of 6-8-week-old female WT and cGAS-/- mice. Once tumors reached ∼50 mm3, mice received a single 30 Gy dose of either FLASH or CONV RT targeted to the tumor site. Tumor growth and skin toxicity (graded 0-5) were measured every other day. Euthanasia was performed upon excessive tumor burden or skin injury. Data were analyzed by two-way ANOVA followed by Tukey’s post hoc test (p < 0.05). Results: Both FLASH and CONV RT significantly reduced tumor volume (p < 0.001), with complete regression by day 14. Tumor recurrence occurred around day 25 in all groups. In WT mice, both modalities produced comparable tumor control, with CONV showing a slight but non-significant trend toward smaller recurrent tumors and few complete responses but caused severe skin toxicity (score 5) requiring euthanasia by day 50-60. In contrast, FLASH treated mice showed equivalent tumor suppression markedly reducing skin toxicity (score ≤ 3 in 2/10 mice). Notably, the cGAS-/- mice exhibited minimal or no visible toxicity with either modality, except one CONV-treated mouse (score 2). Conclusion: FLASH RT significantly reduces normal tissue toxicity compared to CONV RT while maintaining equivalent tumor control: improved tumor control might be achievable through higher FLASH doses because of the improved therapeutic index over CONV RT. The minimal toxicity observed following FLASH RT, together with the absence of toxicity in cGAS-/- mice, also supports the possibility that FLASH may limit early inflammatory responses and tissue injury by suppression or abrogation of cGAS-STING activation. These findings support FLASH RT as a promising, less-toxic radiotherapeutic approach and highlights the potential of suppression of the cGAS-STING pathway to improve treatment outcomes in BC. Citation Format: Banita Verma, Adel Mutahar, Stavros Melemenidis, Rohit Verma, Lucy Whitemore, Suparna Dutt, Kathleen C. Horst, Edward Elliot Graves, Michael F. Clarke, Lingyin Li, Billy W. Loo, Frederick M. Dirbas. Impact of ultra-high dose rate (FLASH) versus conventional radiotherapy on tumor control in wild-type and cGAS-knockout mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5274.

Management of second ipsilateral breast cancer events (iBCEs) remains controversial, and there is a need to collate existing evidence and international guidance on patient selection and local treatment strategies. This project, endorsed by US and European surgical and radiation oncology societies, aimed to gather expert consensus on these issues. A questionnaire on second iBCE local treatment was developed and reviewed by a core group of eight experts, and Delphi methodology was applied over two rounds to 36 panellists, including radiation oncologists, breast surgeons, a plastic surgeon, and medical physicists. Consensus was predefined as agreement of 75% or higher. After two rounds, consensus was reached for 78 (80%) of 97 items. Panellists agreed that patient preferences are central to decision making (100%) and that a second breast-conserving therapy represents a reasonable option for selected patients (100%). Criteria associated with greater suitability for second breast-conserving therapy included an interval between surgeries of at least 60 months, low-risk accelerated partial breast irradiation classification, luminal molecular profile, and no grade 3 late toxicity related to the first breast-conserving therapy. HER2 (also known as ERBB2)-positive or triple negative subtypes were not viewed as absolute contraindications. Strong consensus was also observed regarding the importance of tumour-to-breast volume ratio, clear surgical margins, and tumour bed reirradiation. For patients undergoing mastectomy, immediate autologous reconstruction was preferred (94%) over implant-based approaches (75%). This international Delphi consensus offers structured guidance for the local management of second iBCE and supports shared decision making and individualised treatment planning.

The frequent development of chemoresistance in cancer presents a major clinical challenge, yet the underlying causes of heterogeneous drug responses remain largely elusive. Here, we systematically assessed the cellular differentiation status of human breast cancer cells using single-cell atlases and identified a distinct population of immature basal-like cancer cells marked by BCL11B. Notably, higher levels of BCL11B+ cancer cells are significantly associated with early relapse in patients with breast cancer who received chemotherapy. Functioning as a central regulator, B-cell lymphoma/leukemia 11B (BCL11B) delineates an immature cell state that preferentially transitions to a drug-resistant persister state during treatment through multiple preexisting and adaptive drug resistance programs. The cytokine tumor necrosis factor-α (TNFα) is revealed as a natural inhibitor of BCL11B and can directly reverse the emergence of chemoresistant persister cells. Therefore, we identify BCL11B as an unappreciated predeterminant of drug response and a therapeutic target for a subset of patients with breast cancer at high risk of developing chemoresistance.

Triple-negative breast cancer (TNBC), defined by a lack of hormone receptors and lack of HER2 amplification, is the deadliest subtype of breast cancer. TNBC kills 1 in 3 patients and while promising new drugs based on PARP inhibition and anti-PD1 immunotherapy can extend survival in selected patients, 30-40% of patients relapse or fail therapy. Increasing evidence suggests that TNBC tumors harbor stem-like tumorigenic cancer cells with high plasticity, capable of replenishing cancer cell populations and linked to adverse outcomes. Unfortunately, the phenotypic diversity, microenvironmental contexture, and functional significance of these cells remain poorly understood and available marker genes are not specific, precluding rational drug development. We created a multimodal single-cell RNA-seq atlas of 80 TNBC patients, including clinical covariates such as BRCA1/2 mutations, race/ethnicity, and prior treatment status. We then defined differentiation-associated malignant cell states using a novel interpretable deep learning approach for determining single-cell potency. We subsequently interrogated geospatial features, including localized microenvironments of less differentiated malignant cells, and linked them to immune checkpoint inhibitor (ICI) response. Our quantitative analysis shows that TNBC is enriched in cancer cells with high developmental potency as compared to other breast cancer subtypes, consistent with its aggressive phenotype. Critically, we observed a strong association between enrichment of high potency cells and resistance to ICI treatment across human and mouse bulk expression data, both within TNBC and across other cancer types, a finding that we experimentally validated in a murine model of TNBC. In scRNA-seq and spatial transcriptomics data, we defined striking relationships between less differentiated cancer cells and context-dependent T cell states indicative of strong immunosuppressive capability. In line with this, we found that knockout of genes associated with immature cancer cells promotes TNBC cell killing by T cells in a co-culture CRISPR screen. Ongoing experiments include perturbations of novel therapeutic targets emerging from this work, with the goal of improving immunotherapy responses and ultimately extending patient survival. We anticipate that our approach will expand our understanding of developmental landscapes in TNBC and lead to new opportunities for developing targeted drugs in this devastating disease. Rachel Gleyzer,Farnaz Khameneh,Zhen Qi,Wubing Zhang,Chloé B. Steen,Minji Kang,Maya Maalouf,Melanis Ghadimi,Makenna Lindsey,Sally Bobo,Thomas J. Lomis,Frederick M. Dirbas,Michael F. Clarke,Aaron M. Newman. Decoding developmental programs driving tumorigenesis and immunotherapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 113.

The development of chemoresistance in many cancer patients poses a significant clinical challenge, while the underlying causes for heterogeneous drug responses remain largely elusive. Increasing evidence suggests that tumors exhibit extensive heterogeneity and harbor cancer subpopulations in less differentiated states that are linked to treatment resistance. Therefore, dissecting immature cancer cell states pre-existing in tumors is essential for identifying biomarkers associated with chemoresistance and establishing targeted therapeutic strategies, particularly in breast cancer, where specific biomarkers are largely lacking. We utilized CytoTRACE, a robust computational framework for predicting the cellular differentiation status from single-cell RNA sequencing data, to unveil the immature cancer cell state and its associated markers in single-cell atlases of human breast cancer. The association between the identified immature cell state and patient survival was determined by inferring the proportions of epithelial cancer cells expressing the marker gene in bulk gene expression datasets. The MMTV-PyMT mouse breast cancer model was used to experimentally investigate the role of the immature cancer cell state in conferring chemoresistance. Single-cell profiling was conducted on tumor samples before and after treatment to examine the inherent characteristics of immature cancer cells and their response to drug pressure. The results obtained from mouse tumors were subsequently confirmed using human breast cancer models. We revealed that BCL11B, a zinc finger transcription factor, defines a distinct immature cancer cell state in human breast cancer that displays basal cell features and demonstrates a heterogeneous expression pattern across breast tumors. Breast cancer patients with higher levels of BCL11B+ cancer cells face a significantly increased risk of relapse following chemotherapy. This pre-existing tumor subpopulation exhibits intrinsic fitness advantages and demonstrates a remarkable ability to evolve into a drug-resistant state through various BCL11B-dependent pre-existing and transcriptional adaptation mechanisms. Notably, TNF-α was pinpointed as a natural inhibitor of BCL11B, and treatment with TNF-α significantly increases chemotherapy efficacy by inducing the exit of the drug-resistant cell state. In conclusion, our findings identify BCL11B as a valuable biomarker for predicting chemotherapy resistance and suggest TNF-α combination treatment for breast cancer patients at high risk of developing chemoresistance. Zhen Qi,1 Gunsagar S. Gulati,2 Angera H. Kuo,1 Shaheen S. Sikandar,3 William Hai Dang Ho,1 Dalong Qian,1 Frederick M. Dirbas,1 Aaron M. Newman,1 Shang Cai,4 Michael F. Clarke1. BCL11B predetermines an immature drug-resistant cell state in breast cancer that is vulnerable to TNF-α treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1256.

Introduction: Radiotherapy is effective for breast cancer treatment but often causes undesirable side effects that impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has shown reduced normal tissue toxicity while achieving comparable tumor growth delay compared to conventional dose rate radiotherapy (CONV). This study evaluated whether FLASH could achieve similar tumor control as CONV with tumor eradication as the primary endpoint, in an orthotopic breast cancer model. Methods: Non-metastatic, orthotopic tumors were generated in the left fourth mammary fat pad using the Py117 mammary tumor cell line in syngeneic C57BL/6J mice. Two sequential irradiation studies were performed using FLASH (93-200 Gy/s) and CONV (0.08 Gy/s) electron beams. Single fractions of 20, 25, or 30 Gy were applied to tumors with varying abdominal wall treatment fields (~3.75 or 2.5 mm treatment margin to tumor). Results: Both FLASH and CONV demonstrated comparable efficacy. Small tumors treated with 30 Gy and larger abdominal wall treatment fields appeared to have complete eradication at 30 days but also exhibited the highest skin toxicity, limiting follow-up and preventing confirmation of eradication. Smaller abdominal wall treatment fields reduced skin toxicity and allowed for extended follow-up, which resulted in 75% tumor-free survival at 48 days. Larger tumors showed growth delay but no eradication. Conclusions: In this preclinical, non-metastatic orthotopic breast cancer model, FLASH and CONV demonstrated equivalent tumor control with single-fraction doses of 20, 25, or 30 Gy. Overall, 30 Gy achieved the highest eradication rate but also resulted in the most pronounced skin toxicity.

Background In case of a second ipsilateral breast cancer event (2ndiBCE) after primary breast conserving treatment (BCT), patients are offered salvage mastectomy (SM) or, for selected patients, 2ndBCT. An international survey dedicated to breast cancer survivors investigated patients’ preferences and expectations. Methods An international survey was drafted jointly with breast cancer patient advocates from the United States (n = 6) and Europe (n = 2). The survey was validated with 33 questions divided into four themes: demographic data, oncological outcomes, patient views and financial issues. The survey was sent to breast cancer survivors. All information provided was anonymized. Results From 06/24 to 10/24, 105 patients answered the online survey. The common patient profile was a white (76 %), married (61.5 %), Christian (58.8 %), US-resident (70.2 %) woman aged between 61 and 70 (62.2 %). Long-term outcomes were perceived as being well established for SM (51.5 %) and 2ndBCT (17.1 %). Breast re-irradiation was considered at risk of complications (65.7 %), and 63 % of the patients thought that 2ndBCT provides a more acceptable body self-image. 2ndBCT was expected to give superior cosmetic outcomes than SM with breast reconstruction (55.4 %). Having enough information and ample time to consider the pros and cons of treatment options were very important/important for 99 % and 96.2 %, respectively. Treatment choice was not influenced by financial concerns for 68.3 %. Conclusion Patient perspective is very important in the decision-making process regarding salvage treatments. Physicians must provide clear, timely information which will enable patients to choose the treatment that best meets their expectations.

Background: Congenital arteriovenous malformation (AVM) of the breast is a rare cause of breast mass caused by abnormal vascular proliferation. Due to high risk of bleeding complications, this lesion presents unique peri- and intra-operative challenges. We describe the successful staged management of a breast AVM in the setting of concomitant bilateral ductal carcinoma in situ (DCIS) and Cowden syndrome, a condition associated with high breast cancer risk. Case Description: A 39-year-old female with Cowden syndrome presented with recently-diagnosed bilateral DCIS and a known recurrent 7.7 cm AVM abutting skin of the upper left breast. In order to minimize risk of intraoperative hemorrhage, she underwent successful preoperative embolization of the AVM. This was followed by bilateral skin-sparing mastectomies with implant-based reconstruction. She experienced no postoperative complications and is without evidence of recurrence after 24 months. Conclusions: AVM of the breast is a rare condition that should be considered in the differential diagnosis of breast mass, particularly in patients with signs of hyperdynamic flow on initial evaluation. This case highlights the merits of preoperative imaging in the workup of a breast mass and the benefit of staged treatment of congenital AVM of the breast in a patient with high oncologic risk. Thorough diagnostic evaluation and multidisciplinary collaboration contributed to a successful outcome in this case.

Purpose: Radiotherapy is an effective breast cancer treatment that enhances local tumor control and prolongs overall survival yet is associated with undesirable side effects which can impair quality of life. Ultra-high dose rate radiotherapy (FLASH) has been shown to induce less normal tissue toxicity while producing comparable tumor growth delay in a variety of preclinical tumor models when compared with conventional dose rate radiotherapy (CONV). However, growth delay is not a surrogate for tumor eradication, which is a critical endpoint of cancer therapy, and studies using FLASH in breast cancer are limited. We sought to evaluate whether FLASH produced comparable tumor control to CONV in a breast cancer model with tumor eradication as the primary endpoint. Methods and Materials: 106 cells from the radiation sensitive mammary tumor cell line Py117 were used to create non-metastatic, syngeneic, orthotopic tumors in the left 4th mammary fat pad of C57BL/6J mice (n=67). Tumors were established for two distinct sequential irradiation studies (Rounds 1 and 2), utilizing either large (7.5 mm into the body) or small (5 mm) treatment tumor margins, respectively. For Round 1, mice were divided into groups with either small (20–40 mm3) or large (250–800 mm3) tumors, whereas only small tumors were included in Round 2. Tumors were irradiated with FLASH (93, 192 and 200 Gy/s) or CONV (0.08 Gy/s) using 16.6 MeV FLASH and 15.7 MeV CONV electron beams. Mice in the small tumor cohort were treated with single fractions of 20, 25, or 30 Gy. The larger tumors were treated with a single fraction of 30 Gy. Tumor eradication was determined by palpation and with histology as needed to clarify physical findings. Results: Single fractions of FLASH and CONV demonstrated comparable treatment responses within matched cohorts of small and large tumors. A portion of small tumors treated with single fractions of 20 or 25 Gy were eradicated though most regrew within 2 to 3 weeks. Eradication of small tumors was best seen treated with 30 Gy and a large treatment tumor margin. These mice had no tumor regrowth at 30 days with either FLASH or CONV: however, euthanasia criteria were met at the 30-day time point due to concerns over skin toxicity for both FLASH and CONV groups. Small tumors treated with 30 Gy and a smaller treatment tumor margin had less skin toxicity with 75% of mice remaining tumor free at 48 days. 30 Gy FLASH and CONV applied to larger tumors demonstrated growth delay equally with a partial reduction in size but without tumor eradication. Conclusions: FLASH and CONV produced comparable tumor control in this model of orthotopic, murine breast tumors. Single fractions of 30 Gy with both FLASH and CONV applied to small tumors achieved the highest rates of tumor eradication in particular when delivered with a wider treatment margin. Skin toxicity seen at this dose and in this location could be ameliorated with the use of multiple fractions or different tangents in future studies. Efforts at eradicating larger tumors would require testing higher single fraction doses, multiple fractions, and/or hypofractionated treatment regimens. The equivalent effectiveness between FLASH and CONV in this study of murine breast tumors supports ongoing evaluation of FLASH for use in treating human breast cancer. To this end future efforts at tumor eradication with single fraction FLASH doses with comprehensive evaluation of the toxicity of organs at risk as compared to CONV will be necessary. Additionally, studies of dose-response in a range of tumor volumes with additional breast cancer cell lines and tumors, including human xenografts, along with refined target margins, will guide future studies into the use of FLASH in the adjuvant therapy of primary human breast cancer. ### Competing Interest Statement FMD is the chair of the scientific advisor board of Beyond Cancer, Ltd. and a member of the clinical advisory board of NasoClenz, Silicon Valley Innovations, Inc (SVI). BWL is a co-founder of TibaRay. BWL is a board member of TibaRay. BWL is a consultant on a clinical trial steering committee for Beigene and has received lecture honoraria from Mevion. All other authors declare no conflicts of interest.

This is an executive summary of the most recent American Society for Radiation Oncology (ASTRO) guidelines on use of partial breast irradiation in early-stage breast cancer. In the conscientious pursuit of “right-sizing” the management of patients with early-stage breast cancer, there has been an emphasis on judicious de-escalation of therapy. A component of this paradigm shift is partial breast irradiation (PBI), an approach characterized by targeted radiation therapy (RT) to lumpectomy cavity margins rather than to the whole breast (i.e., whole breast irradiation [WBI]) after breast conservation surgery (BCS). The American Society for Radiation Oncology (ASTRO) recently completed a revision of its evidence-based guidelines for the application of PBI.1 To accomplish this, recent PBI data were reviewed by panel members, including representatives of the American Society for Radiation Oncology (ASTRO), in collaboration with the American Society of Clinical Oncology (ASCO), and the Society of Surgical Oncology (SSO), which provided representatives and peer reviewers. The guideline was approved by the ASTRO Board of Directors and endorsed by the Canadian Association of Radiation Oncology, European Society for Radiotherapy and Oncology, Royal Australian and New Zealand College of Radiologists, and the Society of Surgical Oncology. The recommendations focused on indications for PBI as an alternative to WBI and technical considerations specific to PBI. This editorial provides a summary and comments on the updated ASTRO PBI guidelines, offering insights into the implications of these findings for clinical practice and multidisciplinary decision-making while underscoring technical considerations for optimal incorporation of PBI into patient care.

Rates of contralateral mastectomy (CM) among patients with unilateral breast cancer have been increasing in the United States. In this Society of Surgical Oncology position statement, we review the literature addressing the indications, risks, and benefits of CM since the society's 2017 statement. We held a virtual meeting to outline key topics and then conducted a literature search using PubMed to identify relevant articles. We reviewed the articles and made recommendations based on group consensus. Patients consider CM for many reasons, including concerns regarding the risk of contralateral breast cancer (CBC), desire for improved cosmesis and symmetry, and preferences to avoid ongoing screening, whereas surgeons primarily consider CBC risk when making a recommendation for CM. For patients with a high risk of CBC, CM reduces the risk of new breast cancer, however it is not known to convey an overall survival benefit. Studies evaluating patient satisfaction with CM and reconstruction have yielded mixed results. Imaging with mammography within 12 months before CM is recommended, but routine preoperative breast magnetic resonance imaging is not; there is also no evidence to support routine postmastectomy imaging surveillance. Because the likelihood of identifying an occult malignancy during CM is low, routine sentinel lymph node surgery is not recommended. Data on the rates of postoperative complications are conflicting, and such complications may not be directly related to CM. Adjuvant therapy delays due to complications have not been reported. Surgeons can reduce CM rates by encouraging shared decision making and informed discussions incorporating patient preferences.

TPS2686 Background: Tumor ablation is a minimally invasive technique commonly used to treat solid tumors in the liver, kidney, bone, and lung and is usually based on thermal and nonthermal approaches. Local and in-situ tumor ablation methods demonstrate enhanced anti-tumor immune responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. Nitric oxide (NO) is a colorless gas and a short-lived free radical. It is a ubiquitous, endogenously generated gas implicated in the homeostatic regulation of physiological processes. Preclinical studies evaluating the effect of high concentration exogenously administered NO demonstrated its anti-cancer properties and suggested that it may serve as a potent tumoricidal agent. We have previously shown that treating mouse colon carcinoma (CT26) tumor-bearing mice with ultra-high concentrations of nitric oxide (UNO101) upregulates innate and adaptive immune cells both locally and systemically. Beyond Cancer is currently conducting a first-in-human, first-in-class, Phase 1 safety, feasibility, and preliminary efficacy clinical study of UNO101 at multiple institutions in Israel. Methods: The study is a 2-part Phase 1 trial with a Dose Escalation and a Dose Expansion portion (NCT05351502). A conventional 3+3 dose escalation will evaluate three cohorts of UNO101 single dose: 25,000, 50,000 and 100,000 parts per million (PPM) delivered intratumorally over 5 minutes in subjects with an ECOG PS of 0–3, at least 3 months of life expectancy, with relapsed or refractory unresectable primary or metastatic cutaneous and subcutaneous measurable solid tumors being eligible for enrollment. Upon determination of the maximum tolerated dose or biological effective dose, whichever occurs first, the proposed recommended Phase 2 dose will be further evaluated in the Dose Expansion portion of the study. RECIST version 1.1 and iRECIST will be utilized to assess the rate of malignant tumor response after UNO administration and toxicity will be graded per NCI CTCAE version 5.0. Evaluation of response per itRECIST will also be explored. Up to thirty-eight enrolled subjects are anticipated. Cohort 1, 25,000 PPM have been completed without a reported DLT. Enrollment to Cohort 2, 50,000 PPM, began in January 2024. This study was approved by Israel Ministry of Health (IMOH) as well as the participating institution’s Ethics Board. Written informed consent was obtained for all enrolled subjects and a copy of the written consent is available for review. Clinical trial information: NCT05351502 .

Locoregional recurrence (LRR) of breast cancer represents a heterogenous spectrum of disease. Treatment strategies are tailored to each patient and are impacted by multiple factors. The intent of this review is to discuss recent studies and modern management of isolated LRR. For selected patients with small ipsilateral breast tumor recurrences, partial-breast irradiation following repeat breast-conserving surgery may be appropriate with acceptable rates of toxicities and promising local control. Over the past decade, numerous systemic therapies for the neoadjuvant or adjuvant treatment of early-stage primary breast cancer have been shown to improve oncologic outcomes. Patients with recurrences were not included in these trials yet may benefit from these newer agents. The management of patients with LRR is complex and is influenced by initial treatments, location of recurrence, and hormone and HER-2 receptor status. Patients with isolated LRR have potentially curable disease, and often warrant aggressive multimodality treatment. The addition of systemic therapy remains an area in great need of clinical research.

Fig S1: Similar level of total STAT1/3 and similar level of basal pSTAT1/3 were found in CD4 naïve T cells from healthy donors and BC patients, suggesting that the observed defective IL-6 signaling responses were not caused by lower level of total STAT1/3 or basal pSTAT1/3 in BC patients. Fig S2: Among relapsed BC patients, similar IL-6 signaling responses were found at diagnosis and at relapsed, indicating that impaired IL-6 signaling responses persistent through progression. In relapsed patients who went on to achieve remission, there was a trend towards higher IL-6 signaling in some patients. Fig S3: Lower IL-6 signaling response in peripheral CD4+ naïve T cells from melanoma (Mel), gastrointestinal (GI) and lung cancer (LC) patients. Table S1: No significant associations were found between IL-6 signaling response and clinicopathologic characteristics (age, tumor grade, T status or subtype) of BC patients.

Problem Statement: Radiation therapy (RT) for breast cancer (BC) can induce skin and soft tissue fibrosis, raises concerns over cardiac and pulmonary injury, is associated with higher rates of lymphedema and shoulder dysfunction with regional nodal irradiation, and significantly increases complication rates in women undergoing implant-based reconstruction due to radiation toxicity. Although these toxicities are not generally associated with higher mortality, in general, they can represent significant setbacks with respect to quality of life. These toxicities dissuade some patients from breast conservation leading to unnecessary mastectomy and can lead patients to omit reconstruction after mastectomy or choose more extensive autologous breast reconstruction. Women with implant-based reconstruction and radiotherapy have known higher rates of reconstruction failure. Purpose: Ultra-high dose rate radiation (FLASH) has been shown to induce less normal tissue toxicity, therefore if tumor control of FLASH-RT would be comparable to conventional radiotherapy (CONV) then it has the potential to lower morbidity associated with radiotherapy for breast cancer and allow overall improved outcomes. At first, we aimed to determine the effectiveness of FLASH-RT compared to CONV in eradicating small breast tumors in an orthotopic BC model using single-fraction 20 or 30Gy RT to compare effectiveness of FLASH-RT vs CONV. Methods: Radiation sensitive, syngeneic mammary tumor cell line Py117, that efficiently forms non-metastatic orthotopic tumors in C57BL/6 mice, were injected (106 cells) into the left 4th mammary fat pad. 30mm3 tumors or a range of greater volumes (200-800mm3) were irradiated with single-fraction 20 or 30Gy with a 2x2cm radiation field (~17MeV beams), exposing only 5mm of the surrounding tissue. FLASH RT was delivered with 2Gy per pulse at dose rate ~200Gy/s compared to CONV dose rate of 0.13Gy/s Results: Single-fraction 20Gy suppressed 30mm3 tumor growth until ~day 15 post-RT then regrew for both FLASH and CONV, while 30mm3 tumors were eradicated with both FLASH and CONV at 30Gy. Larger tumors irradiated with 30Gy regressed until ~day 12 post-RT then regrew for both FLASH and CONV. There was no significant difference in growth suppression or tumor eradication between FLASH and CONV in any cohort. Conclusion: In this murine model of breast cancer, FLASH is as effective as CONV in controlling tumor growth. Future studies will extend the evaluation of the tumor control using clinically relevant fractionated dose schedules to be followed by comparisons of tumor control in xenograft models. Additional studies will assess normal tissue toxicity of FLASH vs CONV in murine models of implant-based breast reconstruction. We have established collaborations to understand differences in molecular pathways activated by FLASH vs CONV in tumor and normal tissue to explain the observed experimental differences in normal tissue, tumor, and cancer stem cells. Citation Format: Frederick Dirbas, Stavros Melemenidis, Bill Loo, Kathleen Horst, Edward E. Graves, Suparna Dutt, Vignesh Viswanathan, Brianna Lau, Amy Yu. FLASH-RT, ultra-high dose rate rate radiotherapy, is as effective as conventional dose rate radiotherapy in eradicating tumor in a preclinical model of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-10-02.

Abstract Background: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited to a subset of tumors as response is observed in only ~20% - 50% of patients. Nitric Oxide (NO) is a signaling molecule, found to have a key function in multiple diseases, including cancer. Furthermore, it has been shown to activate anti-tumor immune responses. Previously, we reported that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous NO (UNO) followed by tumor resection stimulated antitumor immune responses. In a previous study, we showed that treatment of 50,000 ppm NO for 10 minutes in combination with anti-mouse-PD-1 (anti-mPD-1) resulted in primary and secondary tumor complete regression in 53% of treated mice at day 100 post-UNO treatment (1). Here, we investigated a higher dose NO over a shorter delivery time to evaluate the hypothesis of improved efficacy of a combination NO and programmed cell death protein-1 (PD-1) antibody in treating CT26 tumor-bearing mice. In this study, we tested the potential efficacy of 100,000 ppm NO administered for 5-minutes in combination with anti-mPD-1. Methods: At day zero, 0.5 X 106 CT26 cells were injected to the right flank of Balb/c male mice (n=8 per group) and at Day three, CT26 cells were injected to the contralateral flank. On Day twelve, tumors (average size 75-100 mm3) were treated intratumorally with UNO (100,000 ppm, 5 minutes, flow rate ~0.2 liter per minute). Day thirteen anti-mPD-1 injections (5mg/kg, q3d, x 5) commenced. Post-treatment tumor volume and survival were monitored thereafter. Results: Complete regression of the primary tumor occurred in 2/8 (25%) of mice treated with combination of 5-minute UNO and anti-mPD-1, 26 days post-treatment. This is compared to 1/8 (12.5%) of controls treated with anti-mPD-1 alone and 0/8 (0%) treated with UNO alone. In the nitrogen + anti-mPD-1 group primary tumor regression occurred in 1/8 mice (12.5%), showing no benefit over anti-mPD-1. Six of eight (~75%) of the mice in the UNO+anti-mPD-1 arm are distant tumor free, compared to 3/8 – 4/8 (37.5% - 50%) in all control arms, resulting in 25% tumor-free mice in the UNO+anti-mPD-1 arm. Survival was increased in the UNO/anti-mPD-1 combination arm compared to anti-mPD-1 alone, 26 days post-treatment. Conclusion: UNO in combination with PD-1 blockade resulted in an increase in the proportion of mice that demonstrate regression of primary tumors, an increase in the number of tumor-free mice, and prolonged survival. Here we present a second study showing similar results at day 26 using a shorter duration of UNO. The results imply that UNO sensitizes CT26 tumors to anti-mPD-1 therapy with improved outcomes compared to each therapy alone. 1. Confino H, Dirbas FM, Goldshtein M, Yarkoni S, Kalaora R, Hatan M, et al. Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect. Cancer Cell Int. 2022;22(1):405. Citation Format: Yana Epshtein, Gavin Choy, Jedidiah M. Monson, Hila Confino, Frederick M. Dirbas, Selena Chaisson. Intratumoral administration of ultra high-concentration Nitric Oxide (UNO) and anti-PD-1 treatment leads to high tumor regression rates and prolonged survival in tumor-bearing mice [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A078.

Background: Immune checkpoint inhibitors have transformed clinical oncology. However, their use is limited as response is observed in only ~20–50% of patients. Previously, we demonstrated that treating CT26 tumor-bearing mice with ultra-high-concentration gaseous nitric oxide (UNO) followed by tumor resection stimulated antitumor immune responses. Accordingly, UNO may improve tumor response to immune checkpoint inhibitors. Here, we investigated the ability of UNO to improve the efficacy of a programmed cell death protein-1 (PD-1) antibody in vitro and in treating CT26 tumor-bearing mice. Methods: CT26 cells were injected into the flank of Balb/c mice (n = 15–16 per group). On day 6, CT26 cells were injected into the contralateral flank, and anti-mPD-1 injections commenced. Primary tumors were treated with intratumoral UNO on day 8. Tumor volume, response rates, toxicity, and survival were monitored. Results: (1) Short exposure to 25,000–100,000 parts per million (ppm) UNO in vitro resulted in significant upregulation of PD-L1 expression on CT26 cells. (2) UNO treatment in vivo consistently reduced cell viability in CT26 tumors. (3) Treatment reduced regulatory T-cell (Treg) levels in the tumor and increased levels of systemic M1 macrophages. UNO responders had increased CD8+ T-cell tumor infiltration. (4) Nine days after treatment, primary tumor growth was significantly lower in the combination arm vs. anti-mPD-1 alone (p = 0.0005). (5) Complete tumor regression occurred in 8/15 (53%) of mice treated with a combination of 10 min UNO and anti-mPD-1, 100 days post-treatment, compared to 4/16 (25%) of controls treated with anti-mPD-1 alone (p = 0.1489). (6) There was no toxicity associated with UNO treatment. (7) Combination treatment showed a trend toward increased survival 100 days post-treatment compared to anti-mPD-1 alone (p = 0.0653). Conclusion: Combining high-concentration NO and immune checkpoint inhibitors warrants further assessment especially in tumors resistant to checkpoint inhibitor therapy.