Gupta, Gaorav P

Abstract Introduction: Hormone receptor positive (HR+), HER2- breast cancer (BC) is the most common subtype of BC. Despite advances in chemotherapy and endocrine therapy optimization, local and systemic recurrences frequently occur, especially for patients with node-positive disease. Recent data suggest a subset of patients with this tumor phenotype may benefit from the addition of anti-Programmed Death-1(aPD1) to neoadjuvant chemotherapy. However, T-cell infiltration (TCI), a biomarker of response to aPD1 and chemotherapy, is typically low in this BC subtype. Preclinical models show that combining preoperative radiation therapy (preop RT) with immune checkpoint inhibitors can enhance TCI and anti-tumor immunity. We conducted the first randomized trial to test whether no-, low- or high-dose RT combined with aPD1 increases TCI and improves responses in non-irradiated, metastatic lymph nodes in HR+/HER2- BC (NCT04443348). Materials & Methods: Eligible patients had ER and/or PR≥10%, HER2 negative invasive BC with clipped and biopsy-proven positive axillary node(s), cT1c-T4c, cN1-3, and at least one of the following criteria: grade 3 or grade 1-2 with cN2-N3, Ki67 >20%, or high genomic assay score. Between 2022-2024, 51 patients from 10 centers were randomized to no (0Gy; control arm), low (9Gy) or high (24Gy) doses of preop RT to the primary tumor with concurrent pembrolizumab followed by a 2-week tumor biopsy. Stratification factors were T1c vs. T2-4 and N1 vs. N2-3. Patients subsequently received pembrolizumab with 12 weeks of paclitaxel, followed by 4 cycles of adriamycin-cyclophosphamide (q2wk or q3wk) with pembrolizumab (200 mg q3 wks or 400 mg q6 wks). Surgery and adjuvant therapy followed standard-of-care. Dual co-primary endpoints were 2wk tumor TCI and nodal pathologic complete response (ypN0) at time of definitive surgery. TCI was assessed by multiplex immunofluorescence (pan-cytokeratin, CD3, CD8), using a rank-based Immunoscore (range 0-1), compared to baseline reference biopsies via two-tailed Mann-Whitney test. ypN0 was evaluated non-comparatively. Secondary endpoints included pCR and Residual Cancer Burden (RCB) 0/1. Results: Among 51 patients enrolled, 49 were evaluable for TCI and 48 for the ypN0 endpoint. Median age was 49.5 years (range 23-78). The cohort had a high-risk clinical profile: 92% had cT2-4, 50% N2-3, and 62% grade 3 tumors. Median ER and PR positivity were 90% (20-100) and 68% (1-98), respectively. Multifocal or multicentric tumors were present in 32%. TCI was assessed at 2 weeks post-treatment. The proportion of tumors with TCI in the upper quartile increased with RT dose escalation: 31% (0Gy), 40% (9Gy), and 53% (24Gy). Median TCI scores at 2 weeks were 0.60 (0Gy), 0.56 (9Gy), and 0.82 (24Gy), compared to a median baseline value of 0.50 from untreated reference samples. A statistically significant increase in TCI was observed only in the 24Gy cohort relative to the untreated group (p=0.027). 70.8% underwent mastectomy, with 39.6% receiving immediate reconstruction. 8% experienced G2/3 wound complications, balanced across arms. Median number of lymph nodes removed was 6.5 (1-42). The overall ypN0 rate was 29%, and 24%(0Gy), 29%(9Gy) and 33%(24Gy) per arm. pCR/RCB 0-1 rates were 18%/27% in the entire cohort and 6%/18%(0Gy), 29%/29%(9Gy), and 19%/33%(24Gy), respectively. Conclusion: The addition of 24Gy preop RT to aPD1 significantly increased tumor TCI in HR+/HER2- early-stage BC. Non-irradiated nodal response rates were promising despite over one-third of the study population with grade 1/2 tumors and high disease burden, laying the foundation for a future randomized trial comparing the efficacy of this novel approach against standard of care for node-positive HR+/HER2- BC. Citation Format: G. Gupta, A. Y. Ho, F. Gharibpoor, Y. Abdou, J. D. Anampa, R. C. Blitzblau, B. C. Calhoun, D. L. Casey, S. F. Dent, L. A. Carey, J. R. Bellon, L. E. Warren, J. Lu, T. J. Hieken, A. G. Taghian, A. Bardia, T. Traina, G. Plitas, K. Gallagher, K. Daugherty, A. Thompson, I. Krop, A. Wolff, E. Mittendorf, J. Fox, H. Garber, E. Hwang, A. J. Khan, J. P. Loene, R. Mutter, S. Patil, C. Santa-Maria, B. V. Vincent, J. L. Wright, L. Spring.Primary Results from the HR+/HER2- Cohort of TBCRC-053 (P-RAD): A Randomized Trial of No, Low, or High Dose Preoperative RADiation with Pembrolizumab and Chemotherapy in Node-Positive, HER2-Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr GS2-05.

TPS632 Background: Male breast cancer is a rare disease, and most cases are hormone receptor-positive (HR+). Due to a lack of clinical trials, male breast cancer has historically been treated based on data extrapolated from women. However, there are substantial knowledge gaps in the comparative efficacy, safety, and patient-reported outcomes of endocrine therapies for male breast cancer. While tamoxifen is the current standard of care, additional data are warranted for aromatase inhibitors (AI), gonadal suppression, and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors. Methods: This is an open-label, multicenter, randomized, phase II trial designed to evaluate different endocrine therapies in men with HR+ and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. A total of 60 men will be enrolled across nine (9) sites within the Translational Breast Cancer Research Consortium (TBCRC). Key eligibility criteria include male sex and stage I, II, or III HR+/HER2- breast cancer before surgical resection of the primary tumor and axillary nodes. Key exclusion criteria include prior anti-cancer therapy for the current breast cancer or any other malignancy within the past 12 months, and inflammatory breast cancer. The trial has two phases. The first phase is a window of opportunity component in which newly diagnosed men are randomized 1:1:1 to either tamoxifen (Arm A), anastrozole (Arm B), or anastrozole plus degarelix (Arm C) given for three weeks. The primary endpoint for the window phase is Ki-67 reduction from the baseline diagnostic biopsy to the research biopsy at the end of the window phase. The second phase consists of neoadjuvant treatment, in which the tamoxifen group is randomized 1:1 to tamoxifen (Arm D) versus tamoxifen plus abemaciclib (Arm E), and the anastrozole alone (Arm B) and anastrozole plus degarelix (Arm C) groups are merged and then randomized 1:1 to anastrozole plus degarelix (Arm F) versus anastrozole plus degarelix plus abemaciclib (Arm G). The duration of the neoadjuvant phase is four (4) months, and the primary endpoint of this phase is residual cancer burden (RCB) index at time of surgery. The trial is powered for the RCB endpoint in a 2 x 2 factorial design to detect a 0.6 unit decrease in RCB index with 80% power and alpha=10%. For the Ki-67 endpoint, we assume an approximately 50% reduction in Arms A and B; Arm C will be of interest if it leads to ≥80% reduction in Ki-67. Secondary endpoints include: estradiol and testosterone levels at baseline, end of window, and during neoadjuvant phase; preoperative endocrine prognostic index (PEPI) score at surgery; adverse events; and patient-reported outcomes (including quality of life). Tumor tissue will be collected for correlative analyses. The study opened to enrollment at Dana-Farber in October 2023, and additional sites will open in 2024. Clinical trial information: NCT05501704 .

Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/one-carbon cycle metabolism contributed to accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1]. To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLq) by ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLq and its proteasomal degradation. Overexpression of POLq in OTK-positive cells resulted in efficient repair of DPC-containing DNA double-strand breaks (DSBs) by POLq-mediated end-joining (TMEJ). Transforming activity of OTKs and other leukemia-inducing oncogenes, especially of those causing inhibition of BRCA1/2 -mediated homologous recombination (HR) with and without concomitant inhibition of DNA-PK -dependent non-homologous end-joining (D-NHEJ), was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLq polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitor or DPC-inducing drug etoposide enhanced anti-leukemia effect of POLq inhibitor (POLqi) in vitro and in vivo. In conclusion, we demonstrated that POLq plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde and that it can be targeted to achieve therapeutic effect.

Abstract Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS4-07.

Abstract Oligometastasis represents an intermediate disease stage between localized and widely metastatic cancer. Efficient identification of patients with oligometastasis remains a barrier for accrual on clinical trials of oligometastasis-directed therapy. Here we review the prospect of circulating tumor DNA–based monitoring to promote sensitive, specific, and cost-efficient detection of cancer recurrence during posttreatment surveillance. Thus, an impetus for the development and implementation of clinical-grade circulating tumor DNA assays should be for the positive impact they will have on clinical investigations of oligometastasis-directed therapy.